Plants Having Improved Growth Characteristics And Method For Making The Same

ABSTRACT

The present invention concerns a method for improving growth characteristics of plants by modulating expression of a nucleic acid sequence encoding a NAP1-like protein. The invention also relates to transgenic plants having improved growth characteristics, which plants have modulated expression of a nucleic acid encoding a NAP1-like protein.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.11/547,395, filed Sep. 29, 2006, which is the national stage application(under 35 U.S.C. 371) of PCT/EP2005/051403 filed Mar. 25, 2005, whichclaims benefit of European application 04101388.9 filed Apr. 2, 2004 andU.S. provisional application 60/563,847 filed Apr. 20, 2004. The entirecontents of each of these applications are hereby incorporated byreference herein.

SUBMISSION OF SEQUENCE LISTING

The Sequence Listing associated with this application is filed inelectronic format via EFS-Web and hereby incorporated by reference intothe specification in its entirety. The name of the text file containingthe Sequence Listing is Sequence_Listing_(—)14546_(—)00061. The size ofthe text file is 63 KB, and the text file was created on Jan. 27, 2010.

The present invention concerns a method for improving growthcharacteristics, and in particular for increasing yield of a plant. Morespecifically, the present invention concerns a method for increasingyield by modulating expression in a plant of a nucleic acid encoding aprotein homologous to the Nucleosome Assembly Protein 1 (NAP1-likeprotein). The present invention also concerns plants having modulatedexpression of a nucleic acid encoding a NAP1-like protein, which plantshave increased yield relative to corresponding wild type plants.

Given the ever-increasing world population, and the dwindling area ofland available for agriculture, it remains a major goal to improve theefficiency of agriculture and to increase the diversity of plants inhorticulture. Conventional means for crop and horticultural improvementsutilise selective breeding techniques to identify plants havingdesirable characteristics. However, such selective breeding techniqueshave several drawbacks, namely that these techniques are typicallylabour intensive and result in plants that often contain heterogeneousgenetic complements that may not always result in the desirable traitbeing passed on from parent plants. Advances in molecular biology haveallowed mankind to manipulate the germplasm of animals and plants.Genetic engineering of plants entails the isolation and manipulation ofgenetic material (typically in the form of DNA or RNA) and thesubsequent introduction of that genetic material into a plant. Suchtechnology has led to the development of plants having various improvedeconomic, agronomic or horticultural traits. Traits of particulareconomic interest are growth characteristics such as high yield.

NAP proteins form a family of related proteins that are known in animalsand are reported to be involved in chromatin-related activities. Thefamily of NAP proteins is characterised by the presence of a conservedsequence known as the NAP domain. The NAP domain is described in thePfam (accession PF00956) and Interpro databases (accession IPR002164).NAP is a component of a multifactor complex that mediates DNA packaginginto nucleosomes (Krude, T. and Keller, C. (2001) Cell. Mol. Life. Sci.58, 665-672). During the S phase of the eukaryotic cell division cycle,newly replicated DNA is rapidly assembled into chromatin. This processrequires the coordinated action of several factors. In the initialstages, CAF1 (chromatin assembly factor 1) binds histone proteins H3 andH4 and directs them to the replication fork via PCNA binding. Subsequentdeposition of histone proteins H2A and H2B is mediated by NAP1 proteins.NAP1 was first described in HeLA cells (von Lindern et al. (1992) Mol.Cell. Biol. 12, 3346-3355) and was later found conserved in alleukaryotes. In addition, NAP proteins are thought to regulate genetranscription and may influence cell differentiation and development.

SET proteins are highly related to NAP proteins and play a role invarious cellular processes in humans. In human cells, SET has been shownto be associated with various CDK-cyclin complexes during the regulationof the cell cycle, such as G2/M transition. SET is a potent inhibitor ofProtein Phosphatase 2A (PP2A) that is involved in several signallingpathways. The inhibitory activity of SET could be attributed to anacidic C-terminal domain (Canela et al. (2003) J. Biol. Chem. 278,1158-1164). Other reports show the involvement of SET in DNA repair andtranscription. SET is part of a complex that has DNA binding and bendingactivities mediated by the chromatin-associated protein HMG2. HMG2facilitates the assembly of nucleoprotein higher-order structures bybending and looping DNA or by stabilizing underwound DNA. HMG2co-precipitates with SET (Fan et al. (2002) Mol. Cell. Biol. 22,2810-2820). SET is also reported to inhibit active DNA demethylation(Cervoni et al. (2002) J. Biol. Chem. 277, 25026-25031). The oncoproteinSet/TAF-I, involved in the inhibition of histone acetylation, alsoinhibits demethylation of ectopically methylated DNA resulting in genesilencing. Set/TAF-I is suggested to play a role in integratingepigenetic states of histones and DNA in gene regulation.

The activity of NAP1 proteins is in part regulated by phosphorylation.It was shown that subcellular localization of NAP1 in Drosophila isdependent on its phosphorylation state, which may be controlled byCasein Kinase II (Rodriguez at al (2000) J. Mol. Biol. 298, 225-238).Mammals are reported to possess several NAP1 proteins, while in yeastthere is only one known NAP1 protein.

Plant NAP1 orthologues remain largely unknown, although NAP1 proteinswere reported from soybean (Yoon et al (1995) Mol. Gen. Genet. 249,465-473), Arabidopsis, tobacco, maize and rice (Dong et al. (2003)Planta 216, 561-570). Phylogenetic analysis of plant NAP1-like genes hasrevealed that there are two subgroups, one related to NAP1 and the otherto the SET protein (FIG. 1). Most likely, later sequence divergence mayhave occurred since the two Arabidopsis, the two maize and the twotobacco sequences cluster together pointing to a more recent geneduplication effect. The Saccharomyces cerevisiae genome contains onlyone NAP-encoding gene, combining the functional properties of both theNAP1 and SET subgroups. Similarly, Template Activating Factor 1 (TAF-I),a homologue of NAP1, combines both PP2a inhibiting activity (Saito etal., Biochem. Biophys. Res. Comm. 259, 471-475, 1999) and chromatinremodelling activity (Kawase et al., Genes Cells 1, 1045-1056, 1996). Itis therefore likely that the plant proteins of the NAP/SET family arelargely redundant in function, particularly in the group of SET proteinswhere a lower degree of divergence is observed compared to the NAPgroup. Furthermore, there is structural evidence that NAP and SETproteins belong to the same family since they share the NAP domain whichis followed by a C-terminal acidic region.

Little is known about the function of NAP1-like proteins in plants,although a role in mitosis and cytokinesis has been proposed (Dong et al2003). The plant orthologues of the NAP1 protein most likely play adifferent role than their animal counterparts. Based on its nuclearlocalisation and on sequence similarities with the mammalian SETprotein, a role in chromatin remodelling may be expected for the plantproteins. Furthermore, the plant NAP/SET group of proteins could beinvolved in the regulation of PP2A in plants. PP2A is one of the majorphosphatases in plants, acting to a large extent on transcriptionfactors and protein kinases, and proposed to regulate activity ofproteins involved in a variety of cellular processes, including cellcycle (Ayaydin et al. (2000) Plant J. 23, 85-96), hormonal actions suchas ABA mediated stomatal movement, germination (Kwak et al. (2002) PlantCell 14, 2849-2861), or auxin transport and root development (Garbers etal 1996 EMBO J. 15, 2115-2124). PP2A is furthermore reported to beinvolved in photosynthesis and light signalling (Sheen (1993) EMBO J.12, 3497-3505) and in nitrogen assimilation (Hirose and Yamaya (1999)Plant Physiology 121, 805-812).

To date, no effects on agronomic traits have been described uponmodulating the expression of NAP1-like proteins in plants. It has nowsurprisingly been found that modulating expression of a nucleic acidencoding a NAP1-like protein in a plant gives plants having improvedgrowth and development, in particular increased yield, more particularlyincreased seed yield when compared to corresponding wild type plants.Therefore according to a first embodiment of the present invention thereis provided a method for improving growth and development of a plant,comprising introducing a genetic modification in a plant and selectingfor modulated expression in this plant of a nucleic acid sequenceencoding a NAP1-like protein. In particular, improved growth anddevelopment is increased yield of a plant, more particularly increasedseed yield; and the modulated expression is increased expression.

The term NAP1-like protein, as defined herein, refers to any proteincomprising a NAP domain and an acidic C-terminal region and having PP2aphosphatase inhibiting activity. The term “NAP domain” as used herein isas defined by the Pfam database by accession number PF00956(http://www.sanger.ac.uk/Software/Pfam/; Bateman et al., Nucleic AcidsResearch 30(1):276-280 (2002), see for example Table 1). Preferably,NAP1-like protein sequences useful in the present invention have a NAPdomain comprising a (T/S)FF(T/N/S/E/D)(W/F)(L/F) signature and/or theconserved amino acid sequence as given in SEQ ID NO:33. More preferablythe NAP domain is as represented by SEQ ID NO: 32. The term “acidicC-terminal region” or “acidic C-terminus” as used herein refers to thecarboxy-terminal end of the protein, which carboxy-terminal end is about20 to 25 amino acids long, of which at least 13 residues are glutamicand/or aspartic acid.

TABLE 1 aExamples of Arabidopsis proteins comprising a NAP1 domain GeneID Pfam profile Position Score e-value SEQ ID NO: at1g18800 PF0095627-224 147.7  2e−40 20, 21 at1g74560 PF00956 31-229 135.0 1.3e−36  1, 2at2g19480 PF00956 52-300 457.4 1.2e−133 26, 27 at5g56950 PF00956 52-300473.2 2.2e−138 28, 29 at4g26110 PF00956 52-301 503.4 1.7e−147 24, 25at3g13782 PF00956 69-311 300.7 1.7e−86  30, 31

Optionally, the NAP1-like protein useful in the methods of the presentinvention has, besides being an inhibitor of PP2a phosphatases, alsochromatin remodelling activities. Methods for measuring inhibition ofPP2a phosphatases are known in the art and comprise, for example, assaysbased on commercially available dye-labelled or fluorochrome-labelledsubstrates or assays based on measurement of radioactivity in TCAsoluble fractions after treatment of [³²P]-labelled histone H1 with PP2aphosphatase (Saito et al. (1999) Biochem. Biophys. Res. Comm. 259,471-475) or of radioactivity released from labelled myelin basic protein(Li et al, J. Biol. Chem. 271, 11059-11062, 1996). An alternative methodfor measuring NAP1-like protein activity is given in Example 6, which isbased on the method described by Ulloa et al. (FEBS Letters 330, 85-89,1993). Chromatin remodelling activities may be assayed in several ways,such as measurement of DNA-binding activity in a gel retardation assay(Fan et al., 2002) or as measurement of histone-binding activity usingELISA (Rodriguez et al. (1997) Genomics 44, 253-265). DNA bendingactivity may be determined in a ligase-mediated circularization assay(Fan et al., 2002) or in a supercoiling assay (Fujii-Nakata et al.(1992) J. Biol. Chem. 267, 20980-20986; Yoon et al. (1995), Mol. Gen.Gen. 249, 465-473).

Preferably, the NAP1-like protein, comprising a NAP domain and an acidicC-terminal region as described above, is of plant origin. The NAP1-likeprotein is preferably from a dicotyledonous plant, preferably from thefamily of Brassicaceae, more preferably from Arabidopsis thaliana, mostpreferably the NAP1-like protein is a protein as represented by SEQ IDNO: 2 or is a homologue, derivative or active fragment thereof, whichhomologues, derivatives or active fragments comprise a NAP domain andthe acidic C-terminus as described above, and which homologues,derivatives or active fragments furthermore have PP2A inhibitingactivity. Preferably the NAP1-like proteins are encoded by a nucleicacid as represented by SEQ ID NO: 1 or a nucleic acid capable ofhybridising therewith.

The term “NAP1-like protein” includes proteins homologous to the proteinpresented in SEQ ID NO: 2. Preferred homologues to be used in themethods of the present invention comprise a NAP domain and an acidicC-terminus as described above. Preferably the homologues have a NAPdomain comprising a (T/S)FF(T/N/S/E/D)(W/F)(L/F) signature and/or theconserved sequence of SEQ ID NO:33, and an acidic C-terminus of 20 to 25residues comprising at least 13 aspartic and/or glutamic acid residues.Additionally, the NAP1 homologues have PP2a inhibiting activity andpossess optionally also chromatin-remodelling activities, which can bemeasured as described above.

Homologues of SEQ ID NO: 2 may be found in various eukaryotic organisms.The closest homologues are generally found in the plant kingdom.Homologues of SEQ ID NO: 2 suitable in the methods of the presentinvention include two tobacco proteins (SEQ ID NO: 7 and 9), a tomatoprotein (SEQ ID NO: 23), an alfalfa protein (SEQ ID NO: 11), theArabidopsis protein represented by SEQ ID NO: 21. Other homologuessuitable for practising the method according to the invention includefor example the Zea mays homologues nfa104 (Accession No. AF384036, SEQID NO: 13) and nfa103 (Accession No. AF384035, SEQ ID NO: 19); or theOryza sativa homologues (SEQ ID NO: 15 and 17).

Methods for the search and identification of NAP1-like homologues wouldbe well within the realm of persons skilled in the art. Such methodscomprise comparison of the sequences represented by SEQ ID NO: 1 or 2,in a computer readable format, with sequences that are available inpublic databases such as MIPS (http://mips.gsf.de/), GenBank(http://www.ncbi.nlm.nih.gov/Genbank/index.html) or EMBL NucleotideSequence Database (http://www.ebi.ac.uk/embl/index.html), usingalgorithms well known in the art for the alignment or comparison ofsequences, such as GAP (Needleman and Wunsch, J. Mol. Biol. 48; 443-453(1970)), BESTFIT (using the local homology algorithm of Smith andWaterman (Advances in Applied Mathematics 2; 482-489 (1981))), BLAST(Altschul, S.F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J., J.Mol. Biol. 215:403-410 (1990)), FASTA and TFASTA (W. R. Pearson and D.J. Lipman Proc. Natl. Acad. Sci. USA 85:2444-2448 (1988)). The softwarefor performing BLAST analysis is publicly available through the NationalCentre for Biotechnology Information. The abovementioned homologues wereidentified using blast default parameters (BLOSUM62 matrix, gap openingpenalty 11 and gap extension penalty 1) and preferably the full lengthsequences are used for analysis. Alternatively, only the NAP1 domain maybe used for comparison, since this domain comprises the major part ofthe protein.

“Homologues” of a NAP1-like protein encompass peptides, oligopeptides,polypeptides, proteins and enzymes having amino acid substitutions,deletions and/or insertions relative to the unmodified protein inquestion and having similar biological and functional activity as theunmodified protein from which they are derived. To produce suchhomologues, amino acids of the protein may be replaced by other aminoacids having similar properties (such as similar hydrophobicity,hydrophilicity, antigenicity, propensity to form or break α-helicalstructures or β-sheet structures). Conservative substitution tables arewell known in the art (see for example Creighton (1984) Proteins. W.H.Freeman and Company).

The homologues useful in the method according to the invention have atleast 50% sequence identity or similarity (functional identity) to theprotein sequence as represented in SEQ ID NO: 2 (GenBank accessionNP_(—)177596), alternatively at least 60% or 70% sequence identity orsimilarity to SEQ ID NO:2. Typically, the homologues have at least 80%sequence identity or similarity, preferably at least 85% sequenceidentity or similarity, more preferably at least 90% sequence identityor similarity, most preferably at least 95%, 96%, 97%, 98% or 99%sequence identity or similarity to SEQ ID NO:2.

Alternatively, the homologues useful in the method according to theinvention have at least 40% sequence identity or similarity (functionalidentity) to the protein sequence as represented in GenBank accessionNP_(—)568844 (SEQ ID NO: 29), alternatively at least 50%, 60% or 70%sequence identity or similarity to SEQ ID NO:29. Typically, thehomologues have at least 80% sequence identity or similarity to SEQ IDNO:29, preferably at least 85% sequence identity or similarity, furtherpreferably at least 90% sequence identity or similarity, most preferablyat least 95%, 96%, 97%, 98% or 99% sequence identity or similarity toSEQ ID NO:29.

The percentage of identity may be calculated starting from full-lengthprotein sequences or with certain (preferably conserved) regions of sucha sequence, using alignment programs based on the Needleman and Wunschalgorithm (such as GAP), using the BLOSUM62 matrix, a gap openingpenalty of 10 and a gap extension penalty of 0.5. For example the NAPdomain as given in SEQ ID NO:32 may be used as query. The identificationof such domains in a protein sequence would be well within the realm ofthe person skilled in the art and involve a computer readable format ofthe nucleic acids used in the methods of the present invention, the useof alignment software programs and the use of publicly availableinformation on protein domains, conserved motifs and boxes. Anintegrated search may be done using the INTERPRO database (Mulder etal., (2003) Nucl. Acids Res. 31, 315-318,http://www.ebi.ac.uk/interpro/scan.html) which combines severaldatabases on protein families, domains and functional sites, such as thePRODOM (Servant et al., (2002) Briefings in Bioinformatics 3, 246-251,http://prodes.toulouse.inra.fr/prodom/2002.1/html/home.php), PIR (Huanget al. (2003) Nucl. Acids Res. 31, 390-392, http://pir.georgetown.edu/)or Pfam (Bateman et al. (2002) Nucl. Acids Res. 30, 276-280,http://pfam.wustl.edu/) databases. Sequence analysis programs designedfor motif searching may be used for identification of conservedfragments, regions and domains as mentioned above. Suitable computerprograms include for example MEME (Bailey and Elkan (1994) Proceedingsof the Second International Conference on Intelligent Systems forMolecular Biology, pp. 28-36, AAAI Press, Menlo Park, Calif.,http://meme.sdsc.edu/meme/website/intro.html).

Homologous proteins may be grouped in “protein families”. A proteinfamily may be defined by functional and sequence similarity analysis,such as, for example, Clustal W. A neighbour-joining tree of theproteins homologous to NAP1-like may be generated by the Clustal Wprogram and gives a good overview of its structural and ancestralrelationship. In a particular embodiment of the present invention, theNAP1-like homologue(s) belong(s) to the same protein family as theprotein corresponding to SEQ ID NO: 2.

In the Arabidopsis genome, two family members of the NAP1-like proteinwere identified (NM_(—)177596 (SEQ ID NO: 2), NP_(—)564063 (SEQ ID NO:21)). Family members of the NAP1-like protein may also be identified inother plants such as rice or other monocotyledonous plants.Advantageously these family members are also useful in the methods ofthe present invention.

Two special forms of homology, orthologous and paralogous, areevolutionary concepts used to describe ancestral relationships of genes.The term “paralogous” relates to homologous genes that result from oneor more gene duplications within the genome of a species. The term“orthologous” relates to homologous genes in different organisms due toancestral relationship of these genes. The term “homologues” as usedherein also encompasses paralogues and orthologues of the proteinsuseful in the methods according to the invention.

Orthologous genes may be identified by querying one or more genedatabases with a query gene of interest, using for example the BLASTprogram. The highest-ranking subject genes that result from the searchare then again subjected to a BLAST analysis, and only those subjectgenes that match again with the query gene are retained as trueorthologous genes. For example, to find a rice orthologue of anArabidopsis thaliana gene, one may perform a BLASTN or TBLASTX analysison a rice database (such as (but not limited to) the Oryza sativaNipponbare database available at the NCBI (http://www.ncbi.nlm.nih.gov)or the genomic sequences of rice (cultivars indica or japonica)). In anext step, the obtained rice sequences are used in a reverse BLASTanalysis using an Arabidopsis database. The results may be furtherrefined when the resulting sequences are analysed with ClustalW andvisualised in a neighbour joining tree. This method may be used toidentify orthologues from many different species.

“Homologues” of a NAP1-like encompass proteins having amino acidsubstitutions, insertions and/or deletions relative to the unmodifiedprotein in question. “Substitutional variants” of a protein are those inwhich at least one residue in an amino acid sequence has been removedand a different residue inserted in its place. Amino acid substitutionsare typically of single residues, but may be clustered depending uponfunctional constraints placed upon the polypeptide; insertions willusually be of the order of about 1 to 10 amino acid residues, anddeletions will range from about 1 to 20 residues. Preferably, amino acidsubstitutions comprise conservative amino acid substitutions.“Insertional variants” of a protein are those in which one or more aminoacid residues are introduced into a predetermined site in a protein.Insertions may comprise amino-terminal and/or carboxy-terminal fusionsas well as intra-sequence insertions of single or multiple amino acids.Generally, insertions within the amino acid sequence will be smallerthan amino- or carboxy-terminal fusions. Examples of amino- orcarboxy-terminal fusion proteins or peptides include the binding domainor activation domain of a transcriptional activator as used in the yeasttwo-hybrid system, phage coat proteins, (histidine)₆-tag, glutathioneS-transferase-tag, protein A, maltose-binding protein, dihydrofolatereductase, Tag·100 epitope, c-myc epitope, FLAG®-epitope, lacZ, CMP(calmodulin-binding peptide), HA epitope, protein C epitope and VSVepitope. “Deletion variants” of a protein are characterised by theremoval of one or more amino acids from the protein. Amino acid variantsof a protein may readily be made using peptide synthetic techniques wellknown in the art, such as solid phase peptide synthesis and the like, orby recombinant DNA manipulations.

Methods for the manipulation of DNA sequences to produce substitution,insertion or deletion variants of a protein are well known in the art.For example, techniques for making substitution mutations atpredetermined sites in DNA are well known to those skilled in the artand include M13 mutagenesis, T7-Gen in vitro mutagenesis (USB,Cleveland, Ohio), QuickChange Site Directed mutagenesis (Stratagene, SanDiego, Calif.), PCR-mediated site-directed mutagenesis or othersite-directed mutagenesis protocols.

The term “derivatives” of a NAP1-like protein refers to peptides,oligopeptides, polypeptides, proteins and enzymes which may comprisesubstitutions, deletions or additions of naturally and non-naturallyoccurring amino acid residues compared to the amino acid sequence of thenaturally-occurring form of the NAP1-like protein (such as the proteinpresented in SEQ ID NO: 2). “Derivatives” of a protein encompasspeptides, oligopeptides, polypeptides, proteins and enzymes which maycomprise naturally occurring altered, glycosylated, acylated ornon-naturally occurring amino acid residues compared to the amino acidsequence of a naturally-occurring form of the polypeptide. A derivativemay also comprise one or more non-amino acid substituents compared tothe amino acid sequence from which it is derived, for example a reportermolecule or other ligand, covalently or non-covalently bound to theamino acid sequence such as, for example, a reporter molecule which isbound to facilitate its detection, and non-naturally occurring aminoacid residues relative to the amino acid sequence of anaturally-occurring protein.

“Active fragments” of a NAP1-like protein encompasses at least theamount of amino acid residues, sufficient to retain similar biologicaland/or functional activity compared to the naturally occurring protein.A preferred active fragment of a NAP1-like protein comprises at least aNAP domain (Pfam 00956) and an acidic C-terminal domain enriched in Dand/or E residues as described above, a more preferred active fragmentfurthermore comprises the conserved sequence of SEQ ID NO:33.

The term NAP1-like nucleic acid/gene, as defined herein, refers to anynucleic acid encoding a NAP1-like protein as defined above, or thecomplement thereof. The nucleic acid may be derived (either directly orindirectly (if subsequently modified)) from any source provided that thenucleic acid, when expressed in a plant, leads to modulated expressionof a NAP1-like nucleic acid/gene or modulated activity and/or levels ofa NAP1-like protein. The nucleic acid may be isolated from a eukaryoticsource, such as yeast or fungi, plants (including algae) or animals(including humans). This nucleic acid may be substantially modified fromits native form in composition and/or genomic environment throughdeliberate human manipulation. The nucleic acid sequence is preferably ahomologous nucleic acid sequence, i.e. a nucleic acid sequence encodinga protein structurally and/or functionally related to SEQ ID NO:2,preferably obtained from a plant, whether from the same plant species ordifferent. Preferably, the nucleic acid is as represented by SEQ ID NO:1 or a portion thereof or a nucleic acid sequence capable of hybridisingtherewith, which hybridising sequence encodes proteins having NAP1-likeprotein activity as described above; or it is a nucleic acid encoding anamino acid represented by SEQ ID NO: 2 or encoding a homologue,derivative or active fragment thereof. This term also encompassesvariants of the nucleic acid encoding a NAP1-like protein due to thedegeneracy of the genetic code; allelic variants; and different splicevariants of the nucleic acid encoding a NAP1-like protein, includingvariants that are interrupted by one or more intervening sequences.

Advantageously, the method according to the present invention may alsobe practised using portions of a nucleic acid sequence encoding aNAP1-like protein as defined above, such as the NAP1-like proteinencoded by SEQ ID NO: 1, or by using sequences that hybridise to anucleic acid sequence encoding a NAP1-like protein as defined above,such as SEQ ID NO: 1, preferably under stringent conditions, (whichhybridising sequences encode proteins having NAP1-like activity), or byusing nucleic acids encoding homologues, derivatives or active fragmentsof a NAP1-like protein, such as the one represented by SEQ ID NO:2.

Portions of a DNA sequence refer to a piece of DNA derived or preparedfrom an original (larger) DNA molecule, which DNA portion, whenexpressed in a plant, gives rise to plants having improved growthcharacteristics. Preferably, the improved growth characteristics areincreased yield, more preferably increased seed yield, most preferablythe increased seed yield comprises one or more of increased harvestindex, increased total weight of seeds and increased number of filledseeds. The portion may comprise many genes, with or without additionalcontrol elements, or may contain just spacer sequences etc.

The present invention also encompasses nucleic acid sequences capable ofhybridising with a nucleic acid sequence encoding a NAP1-like protein,which nucleic acid sequences may also be useful in practising themethods according to the invention. The term “hybridisation” as definedherein is a process wherein substantially homologous complementarynucleotide sequences anneal to each other. The hybridisation process canoccur entirely in solution, i.e. both complementary nucleic acids are insolution. Tools in molecular biology relying on such a process includethe polymerase chain reaction (PCR; and all methods based thereon),subtractive hybridisation, random primer extension, nuclease S1 mapping,primer extension, reverse transcription, cDNA synthesis, differentialdisplay of RNAs, and DNA sequence determination. The hybridisationprocess may also occur with one of the complementary nucleic acidsimmobilised to a matrix such as magnetic beads, Sepharose beads or anyother resin. Tools in molecular biology relying on such a processinclude the isolation of poly (A⁺) mRNA. The hybridisation process mayfurthermore occur with one of the complementary nucleic acidsimmobilised to a solid support such as a nitro-cellulose or nylonmembrane or immobilised by e.g. photolithography to, for example, asiliceous glass support (the latter known as nucleic acid arrays ormicroarrays or as nucleic acid chips). Tools in molecular biologyrelying on such a process include RNA and DNA gel blot analysis, colonyhybridisation, plaque hybridisation, in situ hybridisation and microarray hybridisation. In order to allow hybridisation to occur, thenucleic acid molecules are generally thermally or chemically denaturedto melt a double strand into two single strands and/or to removehairpins or other secondary structures from single stranded nucleicacids. The stringency of hybridisation is influenced by conditions suchas temperature, salt concentration and hybridisation buffer composition.

For applications requiring high selectivity, one will typically desireto employ relatively stringent conditions to form the hybrids, e.g., onewill select relatively low salt and/or high temperature conditions, suchas provided by about 0.02 M to about 0.15 M NaCl at temperatures ofabout 50° C. to about 70° C.

High stringency conditions for hybridisation thus include hightemperature and/or low salt concentration (salts include NaCl andNa₃-citrate) but may also be influenced by the inclusion of formamide inthe hybridisation buffer and/or lowering the concentration of compoundssuch as SDS (sodium dodecyl sulphate) in the hybridisation buffer and/orexclusion of compounds such as dextran sulphate or polyethylene glycol(promoting molecular crowding) from the hybridisation buffer.Sufficiently low stringency hybridisation conditions are particularlypreferred for the isolation of nucleic acids homologous to the DNAsequences useful in the methods of the invention defined supra. Elementscontributing to homology include allelism, degeneration of the geneticcode and differences in preferred codon usage. The presence ofmonovalent cations in the hybridisation solution reduce theelectrostatic repulsion between the two nucleic acid strands therebypromoting hybrid formation; this effect is visible for sodiumconcentrations of up to 0.4M. Formamide reduces the melting temperatureof DNA-DNA and DNA-RNA duplexes with 0.6 to 0.7° C. for each percentformamide, and addition of 50% formamide allows hybridisation to beperformed at 30 to 45° C., though the rate of hybridisation will belowered. Base pair mismatches reduce the hybridisation rate and thethermal stability of the duplexes. On average and for large probes, theT_(m) (temperature under defined ionic strength and pH, at which 50% ofthe target sequence hybridises to a perfectly matched probe) decreasesabout 1° C. per % base mismatch.

“Stringent hybridisation conditions” and “stringent hybridisation washconditions” in the context of nucleic acid hybridisation experimentssuch as Southern and Northern hybridisations are sequence dependent andare different under different environmental parameters. For example,longer sequences hybridise specifically at higher temperatures.Specificity is typically the function of post-hybridisation washes.Critical factors of such washes include the ionic strength andtemperature of the final wash solution.

Generally, stringent conditions are selected to be about 50° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength and pH. The T_(m) is dependent upon the solutionconditions and the base composition of the probe, and may be calculatedusing the following equation:

T_(m)=79.8° C.+(18.5×log [Na⁺])+(58.4° C.×%[G+C])−(820×(#bp induplex)⁻¹)−(0.5×% formamide)

Alternative formula's, depending on the types of hybrids, are known inthe art, for example:

-   -   DNA-DNA hybrids (Meinkoth and Wahl, Anal. Biochem., 138:        267-284, 1984):

T_(m)=81.5° C.+16.6×log [Na⁺]^(a)+0.41×%[G/C^(b)]−500×[L^(c)]⁻¹−0.61×%formamide

-   -   DNA-RNA or RNA-RNA hybrids:

T_(m)=79.8+18.5 (log₁₀[Na⁺]^(a))+0.58 (% G/C^(b))+11.8 (%G/C^(b))²−820/L^(c)

-   -   oligo-DNA or oligo-RNA^(d) hybrids:

For <20 nucleotides: T_(m)=2 (I_(n))

For 20-35 nucleotides: T_(m)=22+1.46 (I_(n))

^(a) or for other monovalent cation, but only accurate in the 0.01-0.4 Mrange.^(b) only accurate for % GC in the 30% to 75% range.^(c) L=length of duplex in base pairs.^(d) Oligo, oligonucleotide; I_(n), effective length of primer=2×(no. ofG/C)+(no. of A/T).

Note: for each 1% formamide, the T_(m) is reduced by about 0.6 to 0.7°C., while the presence of 6M urea reduces the T_(m) by about 30° C.

More preferred stringent conditions are when the temperature is 20° C.below T_(m), and the most preferred stringent conditions are when thetemperature is 10° C. below T_(m). Non-specific binding may also becontrolled using any one of a number of known techniques such as, forexample, blocking the membrane with protein containing solutions,additions of heterologous RNA, DNA, and SDS to the hybridisation buffer,and treatment with Rnase.

Wash conditions are typically performed at or below hybridisationstringency. Generally, suitable stringent conditions for nucleic acidhybridisation assays or gene amplification detection procedures are asset forth above. More or less stringent conditions may also be selected.

For the purposes of defining the level of stringency, reference canconveniently be made to Sambrook et al. (2001) Molecular Cloning: alaboratory manual, 3^(rd) Edition Cold Spring Harbor Laboratory Press,CSH, New York or to Current Protocols in Molecular Biology, John Wiley &Sons, N.Y. (1989). An example of low stringency conditions is4-6×SSC/0.1-0.5% w/v SDS at 37-45° C. for 2-3 hours. Depending on thesource and concentration of the nucleic acid involved in thehybridisation, alternative conditions of stringency may be employed suchas medium stringent conditions. Examples of medium stringent conditionsinclude 1-4×SSC/0.25% w/v SDS at ≧45° C. for 2-3 hours. An example ofhigh stringency conditions includes 0.1-1×SSC/0.1% w/v SDS at 60° C. for1-3 hours. The skilled artisan is aware of various parameters which maybe altered during hybridisation and washing and which will eithermaintain or change the stringency conditions. For example, anotherstringent hybridisation condition is hybridisation at 4×SSC at 65° C.,followed by a washing in 0.1×SSC, at 65° C. for about one hour. Analternative example of stringent hybridisation conditions is in 50%formamide, 4×SSC at 42° C. Still another example of stringent conditionsinclude hybridisation at 62° C. in 6×SSC, 0.05× BLOTTO and washing at2×SSC, 0.1% w/v SDS at 62° C.

The methods according to the present invention may also be practisedusing an alternative splice variant of a nucleic acid sequence encodinga NAP1-like protein. The term “alternative splice variant” as usedherein encompasses variants of a nucleic acid sequence in which selectedintrons and/or exons have been excised, replaced or added. Such variantswill be ones in which the biological activity of the protein remainsunaffected, which can be achieved by selectively retaining functionalsegments of the protein. Such splice variants may be found in nature orcan be manmade. Methods for making such splice variants are well knownin the art. Therefore according to another aspect of the presentinvention, there is provided, a method for improving the growthcharacteristics of plants, comprising modulating expression in a plantof an alternative splice variant of a nucleic acid sequence encoding aNAP1-like protein and/or by modulating activity and/or levels of aNAP1-like protein encoded by the alternative splice variant. Preferably,the splice variant is a splice variant of the sequence represented bySEQ ID NO: 1. Preferably the improved growth characteristics areincreased yield, more preferably increased seed yield, most preferablyincreased seed yield comprises increased harvest index, increased numberof filled seeds and/or increased total weight of seeds.

Advantageously, the methods according to the present invention may alsobe practised using allelic variants of a nucleic acid sequence encodinga NAP1-like protein, preferably an allelic variant of a sequencerepresented by SEQ ID NO: 1. Allelic variants exist in nature andencompassed within the methods of the present invention is the use ofthese natural alleles. Allelic variants encompass Single NucleotidePolymorphisms (SNPs), as well as Small Insertion/Deletion Polymorphisms(INDELs). The size of INDELs is usually less than 100 bp. SNPs andINDELs form the largest NAP1-like of sequence variants in naturallyoccurring polymorphic strains of most organisms. They are helpful inmapping genes and discovery of genes and gene functions. They arefurthermore helpful in identification of genetic loci, e.g. plant genes,involved in determining processes such as growth rate, plant size andplant yield, plant vigor, disease resistance, stress tolerance etc.

The activity of a NAP1-like protein or a homologue thereof may also bemodulated (increased or decreased) by introducing a genetic modification(preferably in the locus of a NAP1-like gene). The locus of a gene asdefined herein is taken to mean a genomic region, which includes thegene of interest and 10 KB up- or downstream of the coding region. Theterm “genetic modification” refers to a change by human intervention inthe genetic content of a cell compared to a wild type cell and includestechniques like genetic engineering, breeding or mutagenesis. The changein genetic content comprises modifications of the genome and includesaddition, deletion and substitution of genetic material in thechromosomes of a plant cell as well as in episomes. The term alsoencompasses the addition of extrachromosomal information to a plantcell. Preferably, the genetic modification results in modulatedexpression of a NAP1-like nucleic acid, more preferably in increasedexpression of a NAP1-like nucleic acid.

The genetic modification may be introduced, for example, by any one (ormore) of the following methods: TDNA activation, TILLING, site-directedmutagenesis, homologous recombination or by introducing and expressingin a plant cell a nucleic acid encoding a NAP1-like protein or ahomologue thereof. Following introduction of the genetic modification,there follows a step of selecting for modulated activity of a NAP1-likeprotein. Preferably the modulated activity of a NAP1-like protein isincreased activity, which increase in activity gives plants havingimproved growth characteristics such as increased seed yield. Theselection step may be based on monitoring the presence or absence ofimproved growth characteristics, or on monitoring the presence orabsence of selectable or screenable marker genes linked an introducednucleic acid of interest.

T-DNA activation tagging (Hayashi et al. Science (1992) 1350-1353)involves insertion of T-DNA usually containing a promoter (may also be atranslation enhancer or an intron), in the genomic region of the gene ofinterest or 10 KB up- or down stream of the coding region of a gene in aconfiguration such that the promoter directs expression of the targetedgene. Typically, regulation of expression of the targeted gene by itsnatural promoter is disrupted and the gene falls under the control ofthe newly introduced promoter. The promoter is typically embedded in aT-DNA. This T-DNA is randomly inserted into the plant genome, forexample, through Agrobacterium infection and leads to overexpression ofgenes near to the inserted T-DNA. The resulting transgenic plants showdominant phenotypes due to overexpression of genes close to theintroduced promoter. The promoter to be introduced may be any promotercapable of directing expression of a gene in the desired organism, inthis case a plant. For example, constitutive, tissue-preferred, celltype-preferred and inducible promoters are all suitable for use in T-DNAactivation.

A genetic modification may also be introduced in the locus of aNAP1-like gene using the technique of TILLING (Targeted Induced LocalLesions IN Genomes). This is a mutagenesis technology useful to generateand/or identify, and to eventually isolate mutagenised variants of aNAP1-like nucleic acid encoding a polypeptide capable of exhibitingNAP1-like activity. TILLING also allows selection of plants carryingsuch mutant variants. These mutant variants may even exhibit higherNAP1-like activity than that exhibited by the gene in its natural form.TILLING combines high-density mutagenesis with high-throughput screeningmethods. The steps typically followed in TILLING are: (a) EMSmutagenesis (Redei and Koncz, 1992; Feldmann et al., 1994; Lightner andCaspar, 1998); (b) DNA preparation and pooling of individuals; (c) PCRamplification of a region of interest; (d) denaturation and annealing toallow formation of heteroduplexes; (e) DHPLC, where the presence of aheteroduplex in a pool is detected as an extra peak in the chromatogram;(f) identification of the mutant individual; and (g) sequencing of themutant PCR product. Methods for TILLING are well known in the art(McCallum Nat. Biotechnol. 2000 April; 18(4):455-7, reviewed by Stemple2004 (TILLING-a high-throughput harvest for functional genomics. Nat RevGenet. 2004 February; 5(2):145-50.)).

Site-directed mutagenesis may be used to generate variants of NAP1-likenucleic acids or portions thereof. Several methods are available toachieve site-directed mutagenesis, the most common being PCR basedmethods (current protocols in molecular biology. Wiley Eds.http://www.4ulr.com/products/currentprotocols/index.html).

TDNA activation, TILLING and site-directed mutagenesis are examples oftechnologies that enable the generation of novel alleles and NAP1-likevariants.

Homologous recombination allows introduction in a genome of a selectednucleic acid at a defined selected position. Homologous recombination isa standard technology used routinely in biological sciences for lowerorganisms such as yeast or the moss Physcomitrella. Methods forperforming homologous recombination in plants have been described notonly for model plants (Offring a et al. Extrachromosomal homologousrecombination and gene targeting in plant cells afterAgrobacterium-mediated transformation. 1990 EMBO J. 1990 October;9(10):3077-84) but also for crop plants, for example rice (Terada R,Urawa H, Inagaki Y, Tsugane K, lida S. Efficient gene targeting byhomologous recombination in rice. Nat. Biotechnol. 2002. Iida andTerada: A tale of two integrations, transgene and T-DNA: gene targetingby homologous recombination in rice. Curr Opin Biotechnol. 2004 April;15(2):132-8). The nucleic acid to be targeted (which may be a NAP1-likenucleic acid or variant thereof as hereinbefore defined) need not betargeted to the locus of a NAP1-like gene, but may be introduced in, forexample, regions of high expression. The nucleic acid to be targeted maybe an improved allele used to replace the endogenous gene or may beintroduced in addition to the endogenous gene.

NAP1-like proteins have a typical domain organisation, consisting of aNAP domain followed by an acidic C-terminal region. Therefore, it isenvisaged that engineering of the domains of the NAP1-like protein insuch a way that the activity of the NAP1-like protein is retained ormodified, is useful for performing the methods of the invention. Apreferred type of variants includes those generated by domain deletion,stacking or DNA shuffling (see for example He et al., Science 288,2360-2363, 2000; or U.S. Pat. Nos. 5,811,238 and 6,395,547), providedthat the resulting NAP1-like protein comprises a NAP domain and anacidic C-terminal region of 20 to 25 amino acids comprising at least 13glutamic and/or aspartic acid residues. Directed evolution may also beused to generate variants of nucleic acids encoding a NAP1-like protein.This consists of iterations of DNA shuffling followed by appropriatescreening and/or selection to generate variants of NAP1-like nucleicacids or portions thereof encoding NAP1-like polypeptides or homologuesor portions thereof having an modified biological activity (Castle etal., (2004) Science 304(5674): 1151-4; U.S. Pat. Nos. 5,811,238 and6,395,547).

Accordingly, as another aspect of the invention, there is provided amethod for improving plant growth and development when compared tocorresponding wild type plants, preferably for increasing plant yield,more preferably for increasing seed yield of a plant, comprisingmodulating expression, preferably increasing expression in a plant of anucleic acid sequence encoding a NAP1-like protein and/or modulatingactivity and/or levels in a plant of a NAP1-like protein, preferablyincreasing activity and/or levels of a NAP1-like protein, wherein thenucleic acid sequence and the proteins include variants chosen from:

-   -   (i) a nucleic acid encoding a NAP1-like protein, wherein the        NAP1-like protein is preferably as represented by SEQ ID NO: 1        or encodes a NAP1-like protein as represented by SEQ ID NO: 2;    -   (ii) an alternative splice variant of a nucleic acid sequence        encoding a NAP1-like protein or wherein said NAP1-like protein        is encoded by a splice variant;    -   (iii) an allelic variant of a nucleic acid sequence encoding a        NAP1-like protein or wherein said NAP1-like protein is encoded        by an allelic variant;    -   (iv) sequence capable of hybridising to a NAP1-like encoding        nucleic acid, preferably under stringent conditions;    -   (v) a NAP1-like protein    -   (vi) a NAP1-like protein as represented by SEQ ID NO: 2    -   (vii) homologues and derivatives of a NAP1-like protein,        preferably of the NAP1-like protein presented in SEQ ID NO:2,    -   and wherein said NAP1-like protein comprises a NAP domain and an        acidic C-terminal, and has PP2a phosphatase inhibiting activity.

Preferably, the increased seed yield comprises one or more of increasedharvest index, increased number of filled seeds and/or increased totalweight of seeds.

In the methods of the present invention modulated expression, and inparticular increased expression, of a nucleic acid is envisaged.Modulating expression (increasing or decreasing expression) of a nucleicacid encoding a NAP1-like protein or modulation of the activity and/orlevels of the NAP1-like protein itself encompasses altered expression ofa gene and/or altered activity and/or levels of a gene product, namely apolypeptide, in specific cells or tissues. Altered (increased ordecreased) expression of a gene and/or altered (increased or decreased)activity and/or levels of a gene product may be effected, for example bychemical means and/or recombinant means. Modulating expression of a geneand/or levels of a gene product and/or modulating activity of a geneproduct may be effected directly through the modulation of expression ofa NAP1-like-encoding gene and/or directly through the modulation of theactivity and/or levels of a NAP1-like protein. The modulated expressionmay result from altered expression levels of an endogenous NAP1-likegene and/or may result from modulated expression of a NAP1-like encodingnucleic acid that was previously introduced into a plant. Similarly,modulated levels and/or activity of a NAP1-like protein may be theresult of altered expression levels of an endogenous NAP1-like geneand/or may result from altered expression of a NAP1-like encodingnucleic acid that was previously introduced into a plant. Additionallyor alternatively, the modulation of expression as mentioned above iseffected in an indirect way, for example it may be effected as a resultof decreased or increased levels and/or activity of factors that controlthe expression of a NAP1-like gene or that influence the activity and/orlevels of the NAP1-like protein.

According to a preferred embodiment of the present invention, modulationof expression of a nucleic acid encoding a NAP1-like protein and/ormodulation of activity and/or levels of the NAP1-like protein itself iseffected by recombinant means. Such recombinant means may comprise adirect and/or indirect approach for modulation of expression of anucleic acid and/or for modulation of the activity and/or levels of aprotein.

A direct and preferred approach for modulating expression of a NAP1-likegene or modulating the activity and/or levels of a NAP1-like protein,comprises introducing into a plant an isolated nucleic acid sequenceencoding a NAP1-like protein or a homologue, derivative or activefragment thereof. The nucleic acid may be introduced into a plant by,for example, transformation. Therefore, according to a preferred aspectof the present invention, there is provided a method for improvinggrowth and development of a plant, in particular for increasing yield ofa plant comprising a genetic modification of the plant, which geneticmodification comprises introducing a NAP1-like encoding nucleic acidinto a plant. Preferably the increased plant yield is increased seedyield, more preferably comprises one or more of increased harvest index,increased number of filled seeds or increased total weight of seeds.

According to a preferred aspect of the present invention, enhanced orincreased expression of a nucleic acid is envisaged. Methods forobtaining enhanced or increased expression of genes or gene products arewell documented in the art and include, for example, overexpressiondriven by a suitable (preferably strong) promoter, the use oftranscription enhancers or translation enhancers. The termoverexpression as used herein means any form of expression that isadditional to the original wild-type expression level. Preferably thenucleic acid to be introduced into the plant and/or the nucleic acidthat is to be overexpressed in the plants is in sense direction withrespect to the promoter to which it is operably linked. Preferably, thenucleic acid to be overexpressed encodes a NAP1-like protein, furtherpreferably the nucleic acid sequence encoding the NAP1-like protein isisolated from a dicotyledonous plant, preferably of the familyBrassicaceae, further preferably the sequence is isolated fromArabidopsis thaliana, most preferably the nucleic acid sequence is asrepresented by SEQ ID NO: 1 or a portion thereof, or encodes an aminoacid sequence as represented by SEQ ID NO: 2 or a homologue, derivativeor active fragment thereof.

Alternatively, the nucleic acid sequence encoding the NAP1-like proteinis as represented in SEQ ID NO: 20 (GenBank Accession NumberNM_(—)101738) or is a portion thereof, or encodes an amino acid sequenceas represented in SEQ ID NO: 21 (GenBank Accession Number NP_(—)564063)or encodes a homologue, derivative or active fragment thereof. It shouldbe noted that the applicability of the invention does not rest upon theuse of the nucleic acid represented by SEQ ID NO: 1, nor upon thenucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 2,but that other nucleic acid sequences encoding homologues, derivativesor active fragments of SEQ ID NO: 2, or portions of SEQ ID NO: 1, orsequences hybridising with SEQ ID NO: 1 may be used in the methods ofthe present invention. In particular, homologues from other species suchas tobacco (SEQ ID NO: 7 or 9), maize (SEQ ID NO: 13 or 19), Medicagosativa (SEQ ID NO: 11), tomato (SEQ ID NO: 23) or rice (SEQ ID NO: 15 or17) are also useful in the methods of the present invention.

The present invention relates to methods to improve growthcharacteristics of a plant or to methods to produce plants with improvedgrowth characteristics, wherein the growth characteristics includeincreased yield, comprising any one or more of: increased number oftillers, increased number of first panicles (being the tallest panicleand all the panicles that overlap with the tallest panicle when alignedvertically), increased number of second panicles, increased total numberof seeds, increased number of filled seeds, increased total seed weightper plant, increased harvest index, increased thousand kernel weight.The present invention also provides methods to improve one of the abovementioned growth characteristics, without causing a penalty on one ofthe other growth characteristics, for example increase of the number offilled seeds while retaining the same number of spikelets per panicle.

The term “increased yield” encompasses an increase in biomass in one ormore parts of a plant relative to the corresponding part(s) of wild-typeplants. The term encompasses an increase in seed yield, which includesan increase in the biomass of the seed (seed weight) and/or an increasein the number of (filled) seeds and/or in the size of the seeds and/oran increase in seed volume, each relative to corresponding wild-typeplants. Depending on the crop, the plant parts in question may beabove-ground biomass (e.g. corn, when used as silage, sugarcane), roots(e.g. sugar beet), fruit (e.g. tomato), cotton fibres, or any other partof the plant which is of economic value. Taking rice as an example, ayield increase may be manifested by an increase in one or more of thefollowing: number of panicles per plant, number of spikelets perpanicle, number of flowers per panicle, increase in the seed fillingrate, increase in thousand kernel weight, among others. For maize, theincrease of seed yield may be reflected in for example an increase ofrows (of seeds) per ear and/or an increased number of kernels per row.An increase in seed size and/or volume may also influence thecomposition of seeds. An increase in seed yield could be due to anincrease in the number and/or size of flowers. An increase in yieldmight also increase the harvest index, which is expressed as a ratio ofthe yield of harvestable parts, such as seeds, over the total biomass;or thousand kernel weight. Increased yield also encompasses the capacityfor planting at higher density (number of plants per hectare or acre).

Also modified cell division may contribute to yield increase. The term“modified cell division” encompasses an increase or decrease in celldivision or an abnormal cell division/cytokinesis, altered plane ofdivision, altered cell polarity, altered cell differentiation. The termalso comprises phenomena such as endomitosis, acytokinesis, polyploidy,polyteny and endoreduplication.

It can be envisaged that plants having increased yield also exhibit amodified growth rate when compared to corresponding wild-type plants.The term “modified growth rate” as used herein encompasses, but is notlimited to, a faster rate of growth in one or more parts of a plant(including seeds), at one or more stages in the life cycle of a plant.Plants with improved growth may show a modified growth curve and mayhave modified values for their T_(mid) or T₉₀ (respectively the timeneeded to reach half of their maximal size or 90% of their maximal size,each relative to corresponding wild-type plants). The term “improvedgrowth” encompasses enhanced vigour, earlier flowering, modified cyclingtime.

According to a preferred feature of the present invention, performanceof the methods according to the present invention results in plantshaving increased yield, in particular plants having increased seedyield. Preferably, the increased seed yield includes at least anincrease in any one or more of number of filled seeds, total seedweight, and harvest index, each relative to control plants. Therefore,according to the present invention, there is provided a method forincreasing yield of plants, which method comprises modulating expressionof a nucleic acid sequence encoding a NAP1-like protein and/ormodulating activity of the NAP1-like protein itself in a plant,preferably wherein the NAP1-like protein is encoded by a nucleic acidsequence represented by SEQ ID NO: 1 or a portion thereof or bysequences capable of hybridising therewith or wherein the NAP1-likeprotein is represented by SEQ ID NO: 2 or is a homologue, derivative oractive fragment thereof. Alternatively, the NAP1-like protein may beencoded by nucleic acid sequences as represented in SEQ ID NO: 20(GenBank Accession Number NM_(—)101738), SEQ ID NO: 6, 8, 10, 12, 14,16, 18 or 22, or by a portion thereof or by sequences capable ofhybridising therewith, or the NAP1-like protein may be as represented inSEQ ID NO: 21 (GenBank Accession Number NP_(—)564063), SEQ ID NO: 7, 9,11, 13, 15, 17, 19, 23 or may be a homologue, derivative or activefragment of any thereof.

The methods of the present invention are favourable to apply to cropplants because the methods of the present invention are used to increaseone or more of the total seed weight, number of filled seeds and harvestindex of a plant. Therefore, the methods of the present invention areparticularly useful for crop plants cultivated for their seeds, such ascereals, sunflower, soybean, pea, flax, lupines, canola. Accordingly, aparticular embodiment of the present invention relates to a method toincrease seed yield (increased total seed weight, increased number offilled seeds and/or to increase harvest index) of a cereal.

According to a further embodiment of the present invention, geneticconstructs and vectors to facilitate introduction and/or expression ofthe nucleotide sequences useful in the methods according to theinvention are provided. Therefore, according to a second embodiment ofthe present invention, there is provided a gene construct for expressionin a plant, comprising:

-   -   (i) a nucleic acid sequence encoding a NAP1-like protein;    -   (ii) one or more control sequences capable of driving expression        in a plant of the nucleic acid sequence of (i); and optionally    -   (iii) a transcription termination sequence.

Constructs useful in the methods according to the present invention maybe created using recombinant DNA technology well known to personsskilled in the art. The gene constructs may be inserted into vectors,which may be commercially available, suitable for transforming intoplants and suitable for expression of the gene of interest in thetransformed cells. The genetic construct may be an expression vectorwherein the nucleic acid sequence is operably linked to one or morecontrol sequences allowing expression in prokaryotic and/or eukaryotichost cells.

According to a preferred embodiment of the invention, the geneticconstruct is an expression vector designed to overexpress the nucleicacid sequence. The nucleic acid sequence capable of modulatingexpression of a nucleic acid encoding a NAP1-like protein and/oractivity of the NAP1-like protein itself may be a nucleic acid sequenceencoding a NAP1-like protein or a homologue, derivative or activefragment thereof, such as any of the nucleic acid sequences describedhereinbefore. A preferred nucleic acid sequence is the sequencerepresented by SEQ ID NO: 1 or a portion thereof or sequences capable ofhybridising therewith or a nucleic acid sequence encoding a proteinrepresented by SEQ ID NO: 2 or a homologue, derivative or activefragment thereof. Preferably, this nucleic acid is cloned in senseorientation relative to the control sequence to which it is operablylinked.

Plants are transformed with a vector comprising the sequence of interest(i.e., the nucleic acid sequence capable of modulating expression ofnucleic acid encoding a NAP1-like protein), which sequence is operablylinked to one or more control sequences (at least a promoter). The terms“regulatory element”, “control sequence” and “promoter” are all usedherein interchangeably and are to be taken in a broad context to referto regulatory nucleic acid sequences capable of effecting expression ofthe sequences to which they are ligated. Encompassed by theaforementioned terms are transcriptional regulatory sequences derivedfrom a classical eukaryotic genomic gene (including the TATA box whichis required for accurate transcription initiation, with or without aCCAAT box sequence) and additional regulatory elements (i.e. upstreamactivating sequences, enhancers and silencers) which alter geneexpression in response to developmental and/or external stimuli, or in atissue-specific manner. Also included within the term is atranscriptional regulatory sequence of a classical prokaryotic gene, inwhich case it may include a −35 box sequence and/or −10 boxtranscriptional regulatory sequences. The term “regulatory element” alsoencompasses a synthetic fusion molecule or derivative which confers,activates or enhances expression of a nucleic acid molecule in a cell,tissue or organ. The term “operably linked” as used herein refers to afunctional linkage between the promoter sequence and the gene ofinterest, such that the promoter sequence is able to initiatetranscription of the gene of interest.

Advantageously, any type of promoter may be used to drive expression ofthe nucleic acid sequence depending on the desired outcome. Preferably,the nucleic acid sequence encoding a NAP1-like protein is operablylinked to a constitutive promoter. The term “constitutive” as definedherein refers to a promoter that is expressed predominantly in at leastone tissue or organ and predominantly at any life stage of the plant.Preferably the promoter is expressed predominantly throughout the plant.Preferably, the constitutive promoter is the GOS2 promoter from rice, ora promoter of similar strength and/or a promoter with a similarexpression pattern. Alternatively, tissue specific promoters may beused. For example, in cases where increased seed yield is envisaged, theuse of seed preferred, flower preferred, meristem preferred promoters orpromoters active in dividing cells can be contemplated. Promoterstrength and/or expression pattern may be analysed for example bycoupling the promoter to a reporter gene and assay the expression of thereporter gene in various tissues of the plant. One suitable reportergene well known to a person skilled in the art is beta-glucuronidase.

Examples of alternative promoters with their respective expressionpattern are presented in Table 2, and these promoters or derivativesthereof may be useful for the methods of the present invention.

TABLE 2 Examples of promoters for use in the performance of the presentinvention Gene source Expresssion pattern Reference Actin constitutiveMcElroy et al, Plant Cell, 2: 163-171, 1990 CAMV 35S constitutive Odellet al, Nature, 313: 810-812, 1985 CaMV 19S constitutive Nilsson et al.,Physiol. Plant. 100: 456-462, 1997 GOS2 constitutive de Pater et al,Plant J Nov; 2(6): 837-44, 1992 ubiquitin constitutive Christensen etal, Plant Mol. Biol. 18: 675-689, 1992 rice cyclophilin constitutiveBuchholz et al, Plant Mol Biol. 25(5): 837-43, 1994 maize H3 histoneconstitutive Lepetit et al, Mol. Gen. Genet. 231: 276-285, 1992 actin 2constitutive An et al, Plant J. 10(1); 107-121, 1996 seed-specific genesseed Simon, et al., Plant Mol. Biol. 5: 191, 1985; Scofield, et al., J.Biol. Chem. 262: 12202, 1987.; Baszczynski, et al., Plant Mol. Biol. 14:633, 1990. Brazil Nut albumin seed Pearson, et al., Plant Mol. Biol. 18:235-245, 1992. legumin seed Ellis, et al., Plant Mol. Biol. 10: 203-214,1988. glutelin (rice) seed Takaiwa, et al., Mol. Gen. Genet. 208: 15-22,1986; Takaiwa, et al., FEBS Letts. 221: 43-47, 1987. zein seed Matzke etal Plant Mol Biol, 14(3): 323-32 1990 napA seed Stalberg, et al, Planta199: 515-519, 1996. wheat LMW and HMW endosperm Mol Gen Genet 216:81-90, 1989; NAR glutenin-1 17: 461-2, 1989 wheat SPA seed Albani et al,Plant Cell, 9: 171-184, 1997 wheat α, β, γ-gliadins endosperm EMBO 3:1409-15, 1984 barley Itr1 promoter endosperm barley B1, C, D, endospermTheor Appl Gen 98: 1253-62, 1999; Plant hordein J 4: 343-55, 1993; MolGen Genet 250: 750-60, 1996 barley DOF endosperm Mena et al, The PlantJournal, 116(1): 53-62, 1998 blz2 endosperm EP99106056.7 syntheticpromoter endosperm Vicente-Carbajosa et al., Plant J. 13: 629-640, 1998.rice prolamin NRP33 endosperm Wu et al, Plant Cell Physiology 39(8)885-889, 1998 rice α-globulin Glb-1 endosperm Wu et al, Plant CellPhysiology 39(8) 885-889, 1998 rice OSH1 embryo Sato et al, Proc. Natl.Acad. Sci. USA, 93: 8117-8122, 1996 rice α-globulin endosperm Nakase etal. Plant Mol. Biol. 33: 513-522, REB/OHP-1 1997 rice ADP-glucose PPendosperm Trans Res 6: 157-68, 1997 maize ESR gene endosperm Plant J 12:235-46, 1997 family sorgum γ-kafirin endosperm PMB 32: 1029-35, 1996KNOX embryo Postma-Haarsma et al, Plant Mol. Biol. 39: 257-71, 1999 riceoleosin embryo and aleuron Wu et at, J. Biochem., 123: 386, 1998sunflower oleosin seed (embryo and dry Cummins, et al., Plant Mol. Biol.19: 873-876, seed) 1992 AtPRP4 flowershttp://salus.medium.edu/mmg/tierney/html chalcone synthase flowers Vander Meer, et al., Plant Mol. Biol. 15, (chsA) 95-109, 1990. LEAFY shootmeristem Weigel et al., Cell 69: 843-859, 1992. Arabidopsis thalianashoot meristem Accession number AJ131822 knat1 Malus domestica kn1 shootmeristem Accession number Z71981 CLAVATA1 shoot meristem Accessionnumber AF049870 Tobacco (N. sylvestris) Dividing cells/ Trehin et al.1997 Plant Mol. Biol. 35, 667-672. cyclin B1; 1 meristematic tissueCatharanthus roseus Dividing cells/ Ito et al. 1997 Plant J. 11, 983-992Mitotic cyclins CYS (A- meristematic tissue type) and CYM (B- type)Arabidopsis cyc1At Dividing cells/ Shaul et al. 1996 (=cyc B1; 1) andmeristematic tissue Proc. Natl. Acad. Sci. U.S.A 93, 4868-4872. cyc3aAt(A-type) Arabidopsis tef1 Dividing cells/ Regad et al. 1995 Mol. Gen.Genet. 248, promoter box meristematic tissue 703-711. Catharanthusroseus Dividing cells/ Ito et al. 1994 Plant Mol. Biol. 24, 863-878.cyc07 meristematic tissue

Optionally, one or more terminator sequences may also be used in theconstruct introduced into a plant. The term “terminator” encompasses acontrol sequence which is a DNA sequence at the end of a transcriptionalunit which signals 3′ processing and polyadenylation of a primarytranscript and termination of transcription. Additional regulatoryelements may include transcriptional as well as translational enhancers.Those skilled in the art will be aware of terminator and enhancersequences which may be suitable for use in performing the invention.Such sequences would be known or may readily be obtained by a personskilled in the art.

The genetic constructs of the invention may further include an origin ofreplication sequence which is required for maintenance and/orreplication in a specific cell type. One example is when a geneticconstruct is required to be maintained in a bacterial cell as anepisomal genetic element (e.g. plasmid or cosmid molecule). Preferredorigins of replication include, but are not limited to, the f1-ori andcolE1.

The genetic construct may optionally comprise a selectable marker gene.As used herein, the term “selectable marker gene” includes any genewhich confers a phenotype on a cell in which it is expressed tofacilitate the identification and/or selection of cells which aretransfected or transformed with a nucleic acid construct of theinvention. Suitable markers may be selected from markers that conferantibiotic or herbicide resistance, that introduce a new metabolic traitor that allow visual selection. Examples of selectable marker genesinclude genes conferring resistance to antibiotics (such as nptII thatphosphorylates neomycin and kanamycin, or hpt, phosphorylatinghygromycin), to herbicides (for example bar which provides resistance toBasta; aroA or gox providing resistance against glyphosate), or genesthat provide a metabolic trait (such as manA that allows plants to usemannose as sole carbon source). Visual marker genes result in theformation of colour (for example β-glucuronidase, GUS), luminescence(such as luciferase) or fluorescence (Green Fluorescent Protein, GFP,and derivatives thereof).

In a preferred embodiment, the genetic construct as mentioned above,comprises a NAP1-like gene in sense orientation coupled to a promoterthat is preferably a constitutive promoter, such as the rice GOS2promoter. Therefore, another aspect of the present invention is a vectorconstruct carrying an expression cassette essentially similar to SEQ IDNO: 3, comprising the rice GOS2 promoter, the Arabidopsis NAP1-like geneand the T-zein+T-rubisco deltaGA transcription terminator sequence. Asequence essentially similar to SEQ ID NO: 3 encompasses a nucleic acidencoding a protein homologous to SEQ ID NO: 2 or hybridising to SEQ IDNO: 1, which nucleic acid is operably linked to a rice GOS2 promoter ora promoter with a similar expression pattern and/or which nucleic acidis linked to a transcription termination sequence.

Therefore according to another aspect of the invention, there isprovided a gene construct, comprising an expression cassette in which islocated a nucleic acid sequence encoding an NAP1-like protein, chosenfrom the group comprising:

-   -   (i) a nucleic acid sequence represented by SEQ ID NO: 1 or the        complement strand thereof;    -   (ii) a nucleic acid sequence encoding an amino acid sequence        represented by SEQ ID NO: 2 or homologues, derivatives or active        fragments thereof;    -   (iii) a nucleic acid sequence capable of hybridising (preferably        under stringent conditions) with a nucleic acid sequence of (i)        or (ii) above, which hybridising sequence preferably encodes a        protein having NAP1-like protein activity;    -   (iv) a nucleic acid sequence according to (i) to (iii) above        which is degenerate as a results of the genetic code;    -   (v) nucleic acid sequence which is an allelic variant to the        nucleic acid sequences according to (i) to (iii);    -   (vi) nucleic acid sequence which is an alternative splice        variant to the nucleic acid sequences according to (i) to (iii);

The present invention also encompasses plants obtainable by the methodsaccording to the present invention. The present invention thereforeprovides plants obtainable by the methods of the present invention,which plants have increased yield and which plants have modulatedNAP1-like protein activity and/or levels and/or modulated expression ofa nucleic acid encoding a NAP1-like protein. Preferably, the plants aretransgenic plants comprising an isolated nucleic acid sequence encodinga NAP1-like protein, characterized in that the transgenic plant has beenselected for having increased yield. Further preferably, the transgenicplant has been selected for modulated expression of a nucleic acidencoding a NAP1-like protein. Furthermore preferably, the transgenicplant has been selected for modulated expression of a nucleic acidencoding a NAP1-like protein as represented by SEQ ID NO:2

According to a third embodiment of the present invention, there isprovided a method for the production of transgenic plants havingimproved growth characteristics, comprising introduction and expressionin a plant of a nucleic acid encoding a NAP1-like protein as describedabove. Preferably, the improved growth characteristics compriseincreased yield, more preferably increased seed yield, most preferablycomprising one or more of increased number of filled seeds, increasedharvest index or increased total weight of seeds.

More specifically, the present invention provides a method for theproduction of transgenic plants having increased yield, which methodcomprises:

-   -   (i) introducing into a plant cell a nucleic acid sequence        encoding a NAP1-like protein;    -   (ii) regenerating and/or growing a plant from a transgenic plant        cell.

The NAP1-like protein itself and/or the NAP1-like nucleic acid itselfmay be introduced directly into a plant cell or into the plant itself(including introduction into a tissue, organ or any other part of theplant). According to a preferred feature of the present invention, thenucleic acid is preferably introduced into a plant by transformation.The nucleic acid is preferably as represented by SEQ ID NO: 1 or aportion thereof or sequences capable of hybridising therewith, or is anucleic acid encoding an amino acid sequence represented by SEQ ID NO: 2or a homologue, derivative or active fragment thereof. Alternatively,the nucleic acid sequence is as represented in SEQ ID NO: 20 (GenBankAccession Number NM_(—)101738), SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 22or a portion thereof or sequences capable of hybridising with any of theaforementioned sequences. The amino acid sequence may alternatively be asequence as represented in SEQ ID NO: 21 (GenBank AccessionNumberNP_(—)564063), SEQ ID NO: 7, 9, 11, 13, 15, 17, 19, 23 orhomologues, derivatives or active fragments thereof.

The term “transformation” as referred to herein encompasses the transferof an exogenous polynucleotide into a host cell, irrespective of themethod used for transfer. Plant tissue capable of subsequent clonalpropagation, whether by organogenesis or embryogenesis, may betransformed with a genetic construct of the present invention and awhole plant regenerated therefrom. The particular tissue chosen willvary depending on the clonal propagation systems available for, and bestsuited to, the particular species being transformed. Exemplary tissuetargets include leaf disks, pollen, embryos, cotyledons, hypocotyls,megagametophytes, callus tissue, existing meristematic tissue (e.g.,apical meristem, axillary buds, and root meristems), and inducedmeristem tissue (e.g., cotyledon meristem and hypocotyl meristem). Thepolynucleotide may be transiently or stably introduced into a host celland may be maintained non-integrated, for example, as a plasmid.Alternatively, it may be integrated into the host genome. The resultingtransformed plant cell can then be used to regenerate a transformedplant in a manner known to persons skilled in the art.

Transformation of a plant species is now a fairly routine technique.Advantageously, any of several transformation methods may be used tointroduce the gene of interest into a suitable ancestor cell.Transformation methods include the use of liposomes, electroporation,chemicals that increase free DNA uptake, injection of the DNA directlyinto the plant, particle gun bombardment, transformation using virusesor pollen and microprojection. Methods may be selected from thecalcium/polyethylene glycol method for protoplasts (Krens, F. A. et al.,1882, Nature 296, 72-74; Negrutiu I. et al., June 1987, Plant Mol. Biol.8, 363-373); electroporation of protoplasts (Shillito R. D. et al., 1985Bio/Technol 3, 1099-1102); microinjection into plant material (CrosswayA. et al., 1986, Mol. Gen. Genet. 202, 179-185); DNA or RNA-coatedparticle bombardment (Klein T. M. et al., 1987, Nature 327, 70)infection with (non-integrative) viruses and the like. Transgenic riceplants expressing an NAP1-like gene are preferably produced viaAgrobacterium-mediated transformation using any of the well knownmethods for rice transformation, such as described in any of thefollowing: European patent application EP 1198985 A1, Aldemita andHodges (Planta, 199, 612-617, 1996); Chan et al. (Plant Mol. Biol. 22(3) 491-506, 1993), Hiei et al. (Plant J. 6 (2) 271-282, 1994), whichdisclosures are incorporated by reference herein as if fully set forth.In the case of corn transformation, the preferred method is as describedin either Ishida et al. (Nat. Biotechnol. 1996 June; 14(6): 745-50) orFrame et al. (Plant Physiol. 2002 May; 129(1): 13-22), which disclosuresare incorporated by reference herein as if fully set forth.

Generally after transformation, plant cells or cell groupings areselected for the presence of one or more markers which are encoded byplant-expressible genes co-transferred with the gene of interest,following which the transformed material is regenerated into a wholeplant.

Following DNA transfer and regeneration, putatively transformed plantsmay be evaluated, for instance using Southern analysis, for the presenceof the gene of interest, copy number and/or genomic organisation.Alternatively or additionally, expression levels of the newly introducedDNA may be monitored using Northern and/or Western analysis, bothtechniques being well known to persons having ordinary skill in the art.

The generated transformed plants may be propagated by a variety ofmeans, such as by clonal propagation or classical breeding techniques.For example, a first generation (or T1) transformed plant may be selfedto give homozygous second generation (or T2) transformants, and the T2plants further propagated through classical breeding techniques.

The generated transformed organisms may take a variety of forms. Forexample, they may be chimeras of transformed cells and non-transformedcells; clonal transformants (e.g., all cells transformed to contain theexpression cassette); grafts of transformed and untransformed tissues(e.g., in plants, a transformed rootstock grafted to an untransformedscion).

The present invention clearly extends to any plant cell or plantproduced by any of the methods described herein, and to all plant partsand propagules thereof. The present invention extends further toencompass the progeny of a primary transformed or transfected cell,tissue, organ or whole plant that has been produced by any of theaforementioned methods, the only requirement being that progeny exhibitthe same genotypic and/or phenotypic characteristic(s) as those producedin the parent by the methods according to the invention. The inventionalso includes host cells containing an isolated nucleic acid moleculeencoding a protein capable of modulating levels and/or activity of aNAP1-like protein, preferably wherein the protein is a NAP1-likeprotein. Preferred host cells according to the invention are plantcells. Therefore, the invention also encompasses host cells ortransgenic plants having altered growth characteristics, characterizedin that the host cell or transgenic plant has modulated expression of anucleic acid sequence encoding a NAP1-like protein and/or modulatedactivity and/or level of a NAP1-like protein. Preferably, the alteredgrowth characteristics comprise increased yield, more preferablyincreased seed yield.

The invention also extends to harvestable parts of a plant such as, butnot limited to, seeds, leaves, fruits, flowers, stems or stem cultures,rhizomes, roots, tubers and bulbs. The invention furthermore relates toproducts directly derived from a harvestable part of such a plant, suchas dry pellets or powders, oil, fat and fatty acids, starch or proteins.

The term “plant” as used herein encompasses whole plants, ancestors andprogeny of the plants and plant parts, plant cells, tissues and organs.The term “plant” also therefore encompasses suspension cultures,embryos, meristematic regions, callus tissue, leaves, flowers, fruits,seeds, roots (including rhizomes and tubers), shoots, bulbs, stems,gametophytes, sporophytes, pollen, and microspores. Plants that areparticularly useful in the methods of the invention include algae,ferns, and all plants which belong to the superfamily Viridiplantae, inparticular monocotyledonous and dicotyledonous plants, including fodderor forage legumes, ornamental plants, food crops, trees, or shrubsselected from the list comprising Abelmoschus spp., Acer spp., Actinidiaspp., Agropyron spp., Allium spp., Amaranthus spp., Ananas comosus,Annona spp., Apium graveolens, Arabidopsis thaliana, Arachis spp,Artocarpus spp., Asparagus officinalis, Avena sativa, Averrhoacarambola, Benincasa hispida, Bertholletia excelsea, Beta vulgaris,Brassica spp., Cadaba farinosa, Camellia sinensis, Canna indica,Capsicum spp., Carica papaya, Carissa macrocarpa, Carthamus tinctorius,Carya spp., Castanea spp., Cichorium endivia, Cinnamomum spp., Citrulluslanatus, Citrus spp., Cocos spp., Coffea spp., Cola spp., Colocasiaesculenta, Corylus spp., Crataegus spp., Cucumis spp., Cucurbita spp.,Cynara spp., Daucus carota, Desmodium spp., Dimocarpus longan, Dioscoreaspp., Diospyros spp., Echinochloa spp., Eleusine coracana, Eriobottyajaponica, Eugenia uniflora, Fagopyrum spp., Fagus spp., Ficus carica,Fortunella spp., Fragaria spp., Ginkgo biloba, Glycine spp., Gossypiumhirsutum, Helianthus spp., Hibiscus spp., Hordeum spp., Ipomoea batatas,Juglans spp., Lactuca sativa, Lathyrus spp., Lemna spp., Lens culinaris,Linum usitatissimum, Litchi chinensis, Lotus spp., Luffa acutangula,Lupinus spp., Macrotyloma spp., Malpighia emarginata, Malus spp., Mammeaamericana, Mangifera indica, Manihot spp., Manilkara zapota, Medicagosativa, Melilotus spp., Mentha spp., Momordica spp., Morus nigra, Musaspp., Nicotiana spp., 0/ea spp., Opuntia spp., Ornithopus spp., Oryzaspp., Panicum miliaceum, Passiflora edulis, Pastinaca sativa, Perseaspp., Petroselinum crispum, Phaseolus spp., Phoenix spp., Physalis spp.,Pinus spp., Pistacia vera, Pisum spp., Poa spp., Populus spp., Prosopisspp., Prunus spp., Psidium spp., Punica granatum, Pyrus communis,Quercus spp., Raphanus sativus, Rheum rhabarbarum, Ribes spp., Rubusspp., Saccharum spp., Sambucus spp., Secale cereale, Sesamum spp.,Solanum spp., Sorghum bicolor, Spinacia spp., Syzygium spp., Tamarindusindica, Theobroma cacao, Trifolium spp., Triticosecale rimpaui, Triticumspp., Vaccinium spp., Vicia spp., Vigna spp., Vitis spp., Zea mays,Zizania palustris, Ziziphus spp., amongst others. According to apreferred feature of the present invention, the plant is a crop plantcomprising soybean, sunflower, canola, alfalfa, rapeseed or cotton.Further preferably, the plant according to the present invention is amonocotyledonous plant such as sugarcane, most preferably a cereal, suchas rice, maize, wheat, millet, barley, oats, sorghum.

The present invention also encompasses the use of nucleic acids encodinga NAP1-like protein, portions or variants thereof and the use ofNAP1-like polypeptides, homologues or derivatives thereof.

One such use relates to improving the growth characteristics of plants,in particular in improving yield, especially seed yield. The seed yieldmay include one or more of the following: increased number of seeds,increased number of filled seeds, increased total seed weight, increasedharvest index, increased thousand kernel weight, seed filling rate,among others. Preferably, the NAP1-like protein or the nucleic acidencoding a NAP1-like protein is of plant origin, more preferably from adicotyledonous plant, furthermore preferably from the family ofBrassicaceae, most preferably, the NAP1-like protein is encoded by SEQID NO:1 or is as represented by SEQ ID NO:2.

NAP1-like nucleic acids or variants thereof or NAP1-like polypeptides orhomologues thereof may find use in breeding programmes in which a DNAmarker is identified that may be genetically linked to a NAP1-like geneor variant thereof. The NAP1-like gene or variants thereof or NAP1-likeprotein or homologues thereof may be used to define a molecular marker.This DNA or protein marker may then be used in breeding programmes toselect plants having improved growth characteristics. The NAP1-like geneor variant thereof may, for example, be a nucleic acid as represented bySEQ ID NO: 1, or a nucleic acid encoding any of the above mentionedhomologues.

Allelic variants of a gene encoding a NAP1-like protein may also finduse in marker-assisted breeding programmes. Such breeding programmessometimes require introduction of allelic variation by mutagenictreatment of the plants, using for example EMS mutagenesis;alternatively, the programme may start with a collection of allelicvariants of so called “natural” origin caused unintentionally.Identification of allelic variants then takes place by, for example,PCR. This is followed by a selection step for selection of superiorallelic variants of the sequence in question and which give rise toimproved growth characteristics in a plant, such as increased seedyield. Selection is typically carried out by monitoring growthperformance of plants containing different allelic variants of thesequence in question, for example, different allelic variants of SEQ IDNO: 1, or of nucleic acids encoding any of the above mentioned planthomologues. Growth performance may be monitored in a greenhouse or inthe field. Further optional steps include crossing plants, in which thesuperior allelic variant resulting in increased seed yield wasidentified, with another plant. This could be used, for example, to makea combination of interesting phenotypic features.

A NAP1-like nucleic acid or variant thereof may also be used as probesfor genetically and physically mapping the genes that they are a partof, and as markers for traits linked to those genes. Such informationmay be useful in plant breeding in order to develop lines with desiredphenotypes. Such use of NAP1-like nucleic acids or variants thereofrequires only a nucleic acid sequence of at least 10 nucleotides inlength. The NAP1-like nucleic acids or variants thereof may be used asrestriction fragment length polymorphism (RFLP) markers. Southern blotsof restriction-digested plant genomic DNA may be probed with theNAP1-like nucleic acids or variants thereof. The resulting bandingpatterns may then be subjected to genetic analyses using computerprograms such as MapMaker (Lander et al. (1987) Genomics 1, 174-181) inorder to construct a genetic map. In addition, the nucleic acids may beused to probe Southern blots containing restriction endonuclease-treatedgenomic DNAs of a set of individuals representing parent and progeny ofa defined genetic cross. Segregation of the DNA polymorphisms is notedand used to calculate the position of the NAP1-like nucleic acid orvariant thereof in the genetic map previously obtained using thispopulation (Botstein et al. (1980) Am. J. Hum. Genet. 32, 314-331).

The production and use of plant gene-derived probes for use in geneticmapping is described in Bematzky and Tanksley (Plant Mol. Biol. Reporter4, 37-41, 1986). Numerous publications describe genetic mapping ofspecific cDNA clones using the methodology outlined above or variationsthereof. For example, F2 intercross populations, backcross populations,randomly mated populations, near isogenic lines, and other sets ofindividuals may be used for mapping. Such methodologies are well knownto those skilled in the art.

The nucleic acid probes may also be used for physical mapping (i.e.,placement of sequences on physical maps; see Hoheisel et al. In:Nonmammalian Genomic Analysis: A Practical Guide, Academic press 1996,pp. 319-346, and references cited therein).

In another embodiment, the nucleic acid probes may be used in directfluorescence in situ hybridization (FISH) mapping (Trask (1991) TrendsGenet. 7, 149-154). Although current methods of FISH mapping favour useof large clones (several to several hundred kb; see Laan et al. (1995)Genome Res. 5, 13-20), improvements in sensitivity may allow performanceof FISH mapping using shorter probes.

A variety of nucleic acid amplification-based methods of genetic andphysical mapping may be carried out using the nucleic acids. Examplesinclude allele-specific amplification (Kazazian (1989) J. Lab. Clin.Med. 11, 95-96), polymorphism of PCR-amplified fragments (CAPS;Sheffield et al. (1993) Genomics 16, 325-332), allele-specific ligation(Landegren et al. (1988) Science 241, 1077-1080), nucleotide extensionreactions (Sokolov (1990) Nucleic Acid Res. 18, 3671), Radiation HybridMapping (Walter et al. (1997) Nat. Genet. 7, 22-28) and Happy Mapping(Dear and Cook (1989) Nucleic Acid Res. 17, 6795-6807). For thesemethods, the sequence of a nucleic acid is used to design and produceprimer pairs for use in the amplification reaction or in primerextension reactions. The design of such primers is well known to thoseskilled in the art. In methods employing PCR-based genetic mapping, itmay be necessary to identify DNA sequence differences between theparents of the mapping cross in the region corresponding to the instantnucleic acid sequence. This, however, is generally not necessary formapping methods.

In this way, generation, identification and/or isolation of modifiedplants with altered NAP1-like activity displaying improved growthcharacteristics can be performed.

NAP1-like nucleic acids or variants thereof or NAP1-like polypeptides orhomologues thereof may also find use as growth regulators. Since thesemolecules have been shown to be useful in improving the growthcharacteristics of plants, they would also be useful growth regulators,such as herbicides or growth stimulators. The present inventiontherefore provides a composition comprising a NAP1-like nucleic acid orvariant thereof or a NAP1-like polypeptide or homologue thereof,together with a suitable carrier, diluent or excipient, for use as agrowth regulator, preferably as a growth promoter, more preferably forincreasing yield.

The methods according to the present invention result in plants havingimproved growth characteristics, as described hereinbefore. Theseadvantageous growth characteristics may also be combined with othereconomically advantageous traits, such as further yield-enhancingtraits, tolerance to various stresses, traits modifying variousarchitectural features and/or biochemical and/or physiological features.Accordingly, the methods of the present invention may also be used inso-called “gene stacking” procedures.

DESCRIPTION OF FIGURES

The present invention will now be described with reference to thefollowing figures in which:

FIG. 1 Phylogenetic tree representing the relationships among NAP andSET proteins from yeast, man and plants. The tree was established by theAlignX program of VNTI Suite 5.5 (Informax,http://www.informaxinc.com/). The matrix used to generate the multiplealignment is Blosum62 and the alingment parameters used were: GapOpening penalty, 10; Gap Extension penalty, 0.5; Gap separation penaltyrange, 8; % identity for alignment delay, 40. The Phylogenetic tree isbuilt using the Neighbor Joining method of Saitou and Nei. GenBank andMIPS (for Arabidopsis thaliana) accession numbers of the sequences usedin the alignment are indicated in the tree. At: Arabidopsis thaliana,Gm: Glycine max, Nt: Nicotiana tabacum (sequences derived from WO03/085115), Os: Oryza sativa, Ps: Pisum sativum, Zm: Zea mays, Hs: Homosapiens, Sc: Saccharomyces cerevisiae.

FIG. 2 Schematic presentation of the entry clone p68, containing CDS0406within the AttL1 and AttL2 sites for Gateway® cloning in the pDONR201backbone. CDS0406 is the internal code for the Arabidopsis NAP1-likecoding sequence. This vector contains also a bacterialkanamycine-resistance cassette and a bacterial origin of replication.

FIG. 3 Binary vector for the expression in Oryza sativa of theArabidopsis NAP1-like gene (CDS0406) under the control of the Gos2promoter (PRO0129). This vector contains a T-DNA derived from the TiPlasmid, limited by a left border (LB repeat, LB Ti C58) and a rightborder (RB repeat, RB Ti C58)). From the left border to the rightborder, this T-DNA contains: a cassette for antibiotic selection oftransformed plants; a selectable marker cassette for visual screening oftransformed plants and the PRO0129-CDS0406-zein and rbcS-deltaGA doubleterminator cassette for expression of the Arabidopsis NAP1-like gene.This vector also contains an origin of replication from pBR322 forbacterial replication and a selectable marker (Spe/SmeR) for bacterialselection with spectinomycin and streptomycin.

FIG. 4 The Medicago NAP1-like protein was expressed in E. coli andpurified from crude cell extract by affinity chromatography via the6×HIS-tag. Elution of the 34 kD protein at different imidazolconcentrations from the nickel-agarose resin is visualised by Westernblotting using anti-6×HIS antibody (Sigma, St Louis, USA).

FIG. 5 Alignment of the Arabidopsis thaliana NAP1-like protein(AtNAP1-like) and the Medicago sativa NAP1-like protein (MsNAP1-like)using the Needleman and Wunsch algorithm. The gap opening penalty wasset on 10, the gap extension penalty was 0.5. With these settings, thesequence identity was 71.2% and sequence homology 84.5%.

FIG. 6 The NAP1-like protein has a nuclear localisation in plants. A)The Medicago NAP1-like protein has been shown to be localised in thenucleus of cultured alfalfa cells by indirect immunofluorescence usingan antibody raised against the purified protein (left picture of panelA). To confirm the nuclear localisation, the nuclei were stained inparallel with the fluorescent dye DAPI, (right picture of panel A). Inthe insert the arrow points to a metaphase cell. A faint fluorescenceindicates low abundance of the NAP1-like protein around the chromosomesin metaphase cells without a nuclear compartment. B) The transientlyexpressed Arabidopsis NAP1-like protein, fused to GFP, is localised tothe nucleus in Arabidopsis cells following a PEG-mediated uptake of thegene construct into protoplasts.

FIG. 7 The purified Medicago NAP1-like protein inhibits in vitrophospho-histone H2B dephosphorylation activity of PP2A (purified fromrabbit skeletal muscle), but has no influence on the dephosphorylationof the glycogen phosphorylase by the same enzyme.

FIG. 8 Examples of sequences useful in performing the methods accordingto the present invention.

EXAMPLES

The present invention will now be described with reference to thefollowing examples, which are by way of illustration alone.

Unless otherwise stated, recombinant DNA techniques are performedaccording to standard protocols described in (Sambrook (2001) MolecularCloning: a laboratory manual, 3rd Edition Cold Spring Harbor LaboratoryPress, CSH, New York) or in Volumes 1 and 2 of Ausubel et al. (CurrentProtocols in Molecular Biology. New York: John Wiley and Sons, 1998).Standard materials and methods for plant molecular work are described inPlant Molecular Biology Labfase (1993) by R. D. D. Croy, published byBIOS Scientific Publications Ltd (UK) and Blackwell ScientificPublications (UK).

Example 1 Gene Cloning

The Arabidopsis NAP1-like (internal reference CDS0406) was amplified byPCR using as template an Arabidopsis thaliana seedling cDNA library(Invitrogen, Paisley, UK). After reverse transcription of RNA extractedfrom seedlings, the cDNAs were cloned into pCMV Sport 6.0. Averageinsert size of the bank was 1.5 kb, and original number of clones was of1.59×10⁷ cfu. Original titer was determined to be 9.6×10⁵ cfu/ml, afterfirst amplification of 6×10¹¹ cfu/ml. After plasmid extraction, 200 ngof template was used in a 50 μl PCR mix. Primers prm1505 (SEQ ID NO: 4)and prm1506 (SEQ ID NO: 5), which include the AttB sites for Gatewayrecombination, were used for PCR amplification. PCR was performed usingHifi Taq DNA polymerase in standard conditions. A PCR fragment of 771 bywas amplified and purified also using standard methods. The first stepof the Gateway procedure, the BP reaction, was then performed, duringwhich the PCR fragment recombines in vivo with the pDONR201 plasmid toproduce, according to the Gateway terminology, an “entry clone”, p68(FIG. 2). Plasmid pDONR201 was purchased from Invitrogen, as part of theGateway® technology.

Example 2 Vector Construction

The entry clone p68 was subsequently used in an LR reaction with p0640,a destination vector used for Oryza sativa transformation. This vectorcontains as functional elements within the T-DNA borders: a plantselectable marker; a visual marker expression cassette; and a Gatewaycassette intended for LR in vivo recombination with the sequence ofinterest already cloned in the entry clone. A GOS2 promoter forconstitutive expression (PRO0129) is located upstream of this Gatewaycassette. After the LR recombination step, the resulting expressionvector p73 (FIG. 3) can be transformed into the Agrobacterium strainLBA4404 and subsequently to Oryza sativa plants.

Example 3 Rice Transformation

Mature dry seeds of Oryza sativa japonica cultivar Nipponbare weredehusked. Sterilization was done by incubating the seeds for one minutein 70% ethanol, followed by 30 minutes in 0.2% HgCl₂ and by 6 washes of15 minutes with sterile distilled water. The sterile seeds were thengerminated on a medium containing 2,4-D (callus induction medium). Aftera 4-week incubation in the dark, embryogenic, scutellum-derived calliwere excised and propagated on the same medium. Two weeks later, thecalli were multiplied or propagated by subculture on the same medium foranother 2 weeks. 3 days before co-cultivation, embryogenic callus pieceswere sub-cultured on fresh medium to boost cell division activity. TheAgrobacterium strain LBA4404 harbouring the binary vector p73 was usedfor co-cultivation. The Agrobacterium strain was cultured for 3 days at28° C. on AB medium with the appropriate antibiotics. The bacteria werethen collected and suspended in liquid co-cultivation medium at an OD₆₀₀of about 1. The suspension was transferred to a petri dish and the calliwere immersed in the suspension during 15 minutes. Next, the callustissues were blotted dry on a filter paper, transferred to solidifiedco-cultivation medium and incubated for 3 days in the dark at 25° C.Thereafter, co-cultivated callus was grown on 2,4-D-containing mediumfor 4 weeks in the dark at 28° C. in the presence of a selective agentat a suitable concentration. During this period, rapidly growingresistant callus islands developed. Upon transfer of this material to aregeneration medium and incubation in the light, the embryogenicpotential was released and shoots developed in the next four to fiveweeks. Shoots were excised from the callus and incubated for 2 to 3weeks on an auxin-containing medium from which they were transferred tosoil. Hardened shoots were grown under high humidity and short days in agreenhouse. Finally seeds were harvested three to five months aftertransplanting. The method yielded single locus transformants at a rateof over 50% (Aldemita and Hodges, Planta 199, 612-617, 1996; Chan etal., Plant Mol. Biol. 22(3), 491-506, 1993; Hiei et al., Plant J. 6(2),271-282, 1994).

Example 4 Evaluation of Transformants: Vegetative Growth Measurements

Approximately 15 to 20 independent TO transformants were generated. Theprimary transformants were transferred from tissue culture chambers to agreenhouse for growing and harvest of T1 seed. Five events of which theT1 progeny segregated 3:1 for presence/absence of the transgene wereretained. For each of these events, 10 T1 seedlings containing thetransgene (hetero- and homo-zygotes), and 10 T1 seedlings lacking thetransgene (nullizygotes), were selected by visual marker screening. Theselected T1 plants were transferred to a greenhouse. Each plant receiveda unique barcode label to link unambiguously the phenotyping data to thecorresponding plant. The selected T1 plants were grown on soil in 10 cmdiameter pots under the following environmental settings:photoperiod=11.5 h, daylight intensity=30,000 lux or more, daytimetemperature=28° C. or higher, night time temperature=22° C., relativehumidity=60-70%. Transgenic plants and the corresponding nullizygoteswere grown side-by-side at random positions. From the stage of sowinguntil the stage of maturity the plants were passed several times througha digital imaging cabinet. At each time point digital images (2048×1536pixels, 16 million colours) were taken of each plant from at least 6different angles.

The mature primary panicles were harvested, bagged, barcode-labelled andthen dried for three days in the oven at 37° C. The panicles were thenthreshed and all the seeds collected. The filled husks were separatedfrom the empty ones using an air-blowing device. After separation, bothseed lots were then counted using a commercially available countingmachine. The empty husks were discarded. The filled husks were weighedon an analytical balance and the cross-sectional area of the seeds wasmeasured using digital imaging. This procedure resulted in the set ofseed-related parameters described below.

These parameters were derived in an automated way from the digitalimages using image analysis software and were analysed statistically. Atwo factor ANOVA (analyses of variance) corrected for the unbalanceddesign was used as statistical model for the overall evaluation of plantphenotypic characteristics. An F-test was carried out on all theparameters measured of all the plants of all the events transformed withthat gene. The F-test was carried out to check for an effect of the geneover all the transformation events and to verify for an overall effectof the gene, also named herein “global gene effect”. If the value of theF test shows that the data are significant, than it is concluded thatthere is a “gene” effect, meaning that not only presence or the positionof the gene is causing the effect. The threshold for significance for atrue global gene effect is set at 5% probability level for the F test.

To check for an effect of the genes within an event, i.e., for aline-specific effect, a t-test was performed within each event usingdata sets from the transgenic plants and the corresponding null plants.“Null plants” or “Null segregants” or “Nullizygotes” are the plantstreated in the same way as the transgenic plant, but from which thetransgene has segregated. Null plants can also be described as thehomozygous negative transformed plants. The threshold for significancefor the t-test is set at 10% probability level. The results for someevents can be above or below this threshold. This is based on thehypothesis that a gene might only have an effect in certain positions inthe genome, and that the occurrence of this position-dependent effect isnot uncommon. This kind of gene effect is also named herein a “lineeffect of the gene”. The p-value is obtained by comparing the t-value tothe t-distribution or alternatively, by comparing the F-value to theF-distribution. The p-value then gives the probability that the nullhypothesis (i.e., that there is no effect of the transgene) is correct.

Vegetative growth and seed yield were measured according to the methodsas described above. The inventors surprisingly found that the totalweight of seeds, the number of filled seeds and the harvest index wereincreased in the rice plants transformed with the NAP1-like gene whencompared the control plants without the NAP1-like gene.

The data obtained in the first experiment were confirmed in a secondexperiment with T2 plants. Four lines that had the correct expressionpattern were selected for further analysis. Seed batches from thepositive plants (both hetero- and homozygotes) in T1, were screened bymonitoring marker expression. For each chosen event, the heterozygoteseed batches were then retained for T2 evaluation. Within each seedbatch an equal number of positive and negative plants were grown in thegreenhouse for evaluation.

A total number of 160 NAP1-like transformed plants were evaluated in theT2 generation, that is 40 plants per event of which 20 positives for thetransgene, and 20 negatives.

Example 5 Evaluation of Transformants: Measurement of Seed-RelatedParameters

Upon analysis of the seeds as described above, the inventors found thatplants transformed with the NAP1-like gene construct had a higher numberof filled seeds, a higher total weight of seeds and an increased harvestindex compared to plants lacking the NAP1-like transgene. Positiveresults obtained for plants in the T1 generation were again obtained inthe T2 generation. Not only individual transgenic lines scoredsignificantly better than the corresponding nullizygous control lines,but there was also a significant positive overall effect when all plantsof all tested T2 events were evaluated, strongly indicating a globalgene effect. An overview of the data is given in Table 3.

TABLE 3 Combined T1 plants T2 plants analyis parameter % increasep-value % increase p-value p-value Total weight of 24 0.0076 28 0.00130.0006 seeds Number of filled 17 0.0352 28 0.0013 0.0003 seeds HarvestIndex 15 0.0085 17 0.0025 0.0003 The % increase presents the averageincrease for all tested events. The p-value stands for the p-valuederived from the F-test.

Number of Filled Seeds

The number of filled seeds was determined by counting the number offilled husks that remained after the separation step. 4 of the 5 testedlines showed an increase in filled seed number, mounting up to 37%.There was an overall increase of 17% in the number of filled seedsproduced by transgenic plants relative to corresponding null segregants,which increase is statistically significant (p-value 0.0352). In the T2generation, there was increase for all tested lines, ranging between 14and 46%. The mean increase for the T2 lines was 28%, this mean increasewas also statistically significant (p-value of 0.0013). The combinedanalysis of T1 and T2 data also confirmed that the global gene effectwas highly significant (p-value of 0.0003).

Total Seed Yield

The total seed yield (total weight of seeds) per plant was measured byweighing all filled husks harvested from a plant. All transgenic T1lines showed an increase in total seed weight, which varied between 8and 43%. On average, the increase in seed yield was 24% and this overalleffect from the presence of the transgene on seed yield was significant,as evidenced by a P-value for the F test of 0.0076. These results werealso observed in the T2 generation. The 4 tested lines had a yieldincrease between 14 and 48% with an average of 28%. This mean increasewas statistically significant (p-value of 0.0013) and also the combinedanalysis of the T1 and T2 plants showed there was a global gene effect(p-value of 0.0006).

Harvest Index

The harvest index in the present invention is defined as the ratiobetween the total seed yield and the above ground area (mm²), multipliedby a factor 10⁶. 4 of the 5 tested lines showed an increased harvestindex, ranging between 9 and 48%. There was a significant overall geneeffect (an effect associated with of the presence of the transgene) onharvest index (an overall increase of 15%), with a statisticallysignificant p-value for the F test of 0.085. Similar results wereobtained for T2 plants. The harvest index for the individual lines wasincreased between 12 and 34% with a significant mean of 17% (p-value of0.0025). Here too, the combined analysis of the T1 and T2 data showed aglobal gene effect (p-value 0.003).

It is known to persons skilled in the art that the expression oftransgenes in plants, and hence also the phenotypic effect due toexpression of such transgene, can differ among different independentlyobtained transgenic lines and progeny thereof. The transgenes present indifferent independently obtained transgenic plants differ from eachother by the chromosomal insertion locus as well as by the number oftransgene copies inserted in that locus and the configuration of thosetransgene copies in that locus. Differences in expression levels can beascribed to influence from the chromosomal context of the transgene (theso-called position effect) or from silencing mechanisms triggered bycertain transgene configurations (e.g. inwards facing tandem insertionsof transgenes are prone to silencing at the transcriptional orpost-transcriptional level). Notwithstanding these possible causes ofvariation, the data show that transgenic plants expressing the NAP1-likegene consistently gave a higher number of filled seeds, a higher totalweight of seeds, as well as an increased Harvest Index, each relative tocorresponding non-transgenic plants. The observed increases weresignificant in both T1 and T2 generation, which is a strong argument fora global gene effect as evidenced by the p-values of the combinedanalysis.

Example 6 Characterisation of a Medicago sativa NAP1-Like ProteinMaterials and Methods

Isolation of the Full-Length cDNA Clone of the Putative AlfalfaPP2A-Inhibitor

An isolated cDNA fragment coding for a part of an alfalfa (Medicagosativa) putative NAP1-like protein has been used to isolate thefull-length clone from an alfalfa root-nodule λ-ZAP phage cDNA library(Savoure et al. Plant Mol. Biol. 27, 1059-1070; 1995) using standardscreening procedures as described by the manufacturer (Stratagene). 400000 plaques were screened, 20 clones were retained, of which 18 werepositive in the second hybridization screen. 8 of these clones wereselected for further work and converted into phagemids from individualphages. Four clones were sequenced and two of them proved to be thefull-length cDNA clones of the putative NAP1-like protein. One of theclones (Ms10.1) was used for further work (SEQ ID NO: 10, encoding theprotein of SEQ ID NO: 11).

Production and Purification of the Medicago NAP1-Like Protein

The cDNA sequence coding for the Medicago sativa NAP1-like protein wasinserted into the NcoI/XhoI site of the pENTRY4 GATEWAY® vector(Invitrogen) and subsequently introduced into the pDEST17 bacterialexpression vector. The pDEST17 vector allowed the expression of theNAP1-like protein in BL21 E. coli cells as a 6×HIS-tagged protein. The34 kDa NAP1-like protein was purified by affinity chromatography using anickel agarose resin (Sigma) (FIG. 4).

Phosphatase Activity Measurements

Potential phosphatase-inhibiting activity of the Medicago sativaNAP1-like protein was tested in vitro on Protein Phosphatase 2A (PP2A)catalytic subunits purified from rabbit skeletal muscle using³²P-isotope-labelled glycogen phosphorylase and histone H2A proteins assubstrates according to Ulloa et al. (1993).

Intracellular Localization of the MsNAP1-Like and AtNAP1-Like Proteins

Polyclonal anti-MsNAP1-like antibodies were raised in rabbits againstthe purified 6×HIS-tagged protein using a standard immunizationprotocol.

Protoplasts were isolated from suspension cultured alfalfa (Medicagosativa) cells and fixed by 6% formaldehyde. The cells were than attachedto poly-L-lysine coated glass slides and exposed to the anti-MsNAP1-likeantiserum (200× diluted in PBS), washed and exposed to FITC-conjugatedgoat anti-rabbit secondary antibody (SIGMA, 100× dilution). Nuclei werestained with DAPI (0.02 mg/ml) in parallel and photographed with a NikonTE300 fluorescent microscope and a SPOT II colour CCD camera.

The coding region of the Arabidopsis thaliana orthologue of the Medicagosativa NAP1-like protein (SEQ ID NO: 1), was inserted in frame with thegreen fluorescent protein (GFP) into the GATEWAY®-compatible plantexpression vector (pK7WGF2). Protoplasts were isolated and transfectedwith the purified plasmid DNA using standard procedures. Transientexpression was recorded one or two days after transfection byfluorescence microscopy.

Results Arabidopsis and Medicado NAP1-Like Proteins are Localised in theNucleus

The NAP1-like protein of Medicago sativa was highly homologous toArabidopsis NAP1-like protein: the sequence identity was 71.2% andsequence similarity was 84.5% (FIG. 5). Using the anti-MsNAP1-likeantibodies, indirect immunofluorescence revealed that the antibodiesrecognised a protein that was localized to the nuclei of suspensioncultured alfalfa cells. This localisation was verified by the nuclearstain, DAPI. Faint fluorescence was associated with the chromosomes inmetaphase cells (FIG. 6.A, insert).

The GFP-tagged Arabidopsis NAP1-like_protein was also exclusivelylocalised to the nuclei of suspension cultured Arabidopsis cells (FIG.6.B).

2) The Alfalfa NAP1-Like Protein Inhibits In Vitro PP2A PhosphataseActivity on a Phospho-Histone Substrate

Purified alfalfa NAP1-like_protein was added at various concentrationsto reaction mixtures containing the catalytic subunits of rabbitskeletal muscle PP2A and phosphorylated histone H2A, or glycogenphosphorylase as substrate. It was observed that the NAP1-like_proteinhad no influence on the dephosphorylation of the glycogen phosphorylaseeven at 500 mM concentration, but already 2.5 mM concentration of theNAP1-like_protein efficiently inhibited PP2A activity on thephospho-histonH2A substrate (50% decrease in activity) (FIG. 7).

CONCLUSION

The Medicago sativa and Arabidopsis thaliana NAP1-like proteins showboth structurally and functionally resemblance. Plant NAP1-like_proteinsinhibit in vitro phosphatase (PP2A) activity on histone substrates,indicating a possible in vivo role on chromatin organisation and genetranscription.

1. A method for improving growth characteristics of a plant relative tocorresponding wild type plants, comprising introducing a geneticmodification in said plant and selecting for modulated expression insaid plant of a nucleic acid sequence encoding a NAP1-like protein. 2.The method according to claim 1, wherein said modulated expression isincreased expression.
 3. The method according claim 1, wherein saidgenetic modification is effected by one of site-directed mutagenesis,homologous recombination, TILLING, T-DNA activation and DNA shuffling.4. The method according to claim 1, wherein said genetic modificationcomprises introducing an isolated nucleic acid sequence encoding aNAP1-like protein into a plant.
 5. The method according to claim 4,wherein said nucleic acid is derived from a plant, preferably from adicotyledonous plant, more preferably from the family Brassicaceae, mostpreferably from Arabidopsis thaliana.
 6. The method according to claim4, wherein said nucleic acid sequence and said proteins include variantschosen from: (i) a nucleic acid encoding a NAP1-like protein, whereinthe NAP1-like protein is preferably as represented by SEQ ID NO: 1 orencodes a NAP1-like protein as represented by SEQ ID NO: 2; (ii) analternative splice variant of a nucleic acid sequence encoding aNAP1-like protein or wherein said NAP1-like protein is encoded by asplice variant; (iii) an allelic variant of a nucleic acid sequenceencoding a NAP1-like protein or wherein said NAP1-like protein isencoded by an allelic variant; (iv) a sequence capable of hybridising toa NAP1-like encoding nucleic acid, preferably under stringentconditions; (v) a NAP1-like protein; (vi) a NAP1-like protein asrepresented by SEQ ID NO: 2; and (vii) homologues and derivatives of aNAP1-like protein, preferably of the NAP1-like protein presented in SEQID NO:2, and wherein said NAP1-like protein comprises a NAP domain andan acidic C-terminal, and has PP2a phosphatase inhibiting activity. 7.The method according to claim 1, wherein said improved growthcharacteristics is increased yield, preferably increased seed yield. 8.The method according to claim 7, wherein said increased seed yieldcomprises one or more of increased number of filled seeds, increasedtotal weight of seeds or increased Harvest Index, each relative tocorresponding wild type plants.
 9. The method according to claim 4,wherein said nucleic acid encoding a NAP1-like protein is operablylinked to a constitutive promoter, preferably a GOS2 promoter.
 10. Amethod for production of a transgenic plant having increased yield,which method comprises: (i) introducing into a plant cell a nucleic acidsequence encoding a NAP1-like protein; and (ii) regenerating and/orgrowing a plant from the transgenic plant cell.
 11. Plants withincreased yield obtainable by the method according to claim 1, includingharvestable parts, propagules, seeds or progeny thereof, and includingproducts directly derived thereof.
 12. A gene construct for expressionin a plant, comprising: (i) a nucleic acid sequence encoding a NAP1-likeprotein; (ii) one or more control sequences capable of drivingexpression in a plant of the nucleic acid sequence of (i); andoptionally (iii) a transcription termination sequence.
 13. The geneconstruct according to claim 12, wherein said nucleic acid encoding aNAP1-like protein is chosen from the group comprising: a nucleic acidsequence represented by SEQ ID NO: 1 or the complement strand thereof;(ii) a nucleic acid sequence encoding an amino acid sequence representedby SEQ ID NO: 2 or homologues, derivatives or active fragments thereof;(iii) a nucleic acid sequence capable of hybridising (preferably understringent conditions) with a nucleic acid sequence of (i) or (ii) above,which hybridising sequence preferably encodes a protein having NAP1-likeprotein activity; (iv) a nucleic acid sequence according to (i) to (iii)above which is degenerate as a results of the genetic code; (v) nucleicacid sequence which is an allelic variant to the nucleic acid sequencesaccording to (i) to (iii); and (vi) nucleic acid sequence which is analternative splice variant to the nucleic acid sequences according to(i) to (iii), and wherein said NAP1-like protein comprises a NAP domainand an acidic C-terminal, and has PP2a phosphatase inhibiting activity.14. The gene construct according to claim 12, wherein said controlsequences comprise at least a constitutive promoter, preferably a GOS2promoter.
 15. The gene construct according to claim 12, wherein saidnucleic acid sequence encoding a NAP1-like protein is oriented in sensedirection relative to said control sequence.
 16. A construct comprisingan expression cassette essentially similar to SEQ ID NO:
 3. 17. Atransgenic plant comprising an isolated nucleic acid sequence encoding aNAP1-like protein, characterized in that said plant has been selectedfor having increased yield.
 18. The transgenic plant of claim 17,selected for increased expression of a nucleic acid encoding a NAP1-likeprotein.
 19. The transgenic plant of claim 17, wherein said isolatednucleic acid encoding a NAP1-like protein encodes a protein asrepresented in SEQ ID NO:2
 20. The transgenic plant according to claim17, wherein said plant is a crop plant comprising soybean, sunflower,canola, alfalfa, rapeseed or cotton, preferably, the plant is amonocotyledonous plant such as sugarcane, most preferably a cereal, suchas rice, maize, wheat, millet, barley, oats, sorghum.
 21. Transgenicplant cells, transgenic plants or transgenic plant parts, includingharvestable parts, propagules, seeds or transgenic progeny, and productsdirectly derived thereof, of a plant according to claim
 17. 22. A methodfor increasing yield of a plant comprising introducing into a plant anucleic acid sequence encoding a NAP1-like protein, or a portion thereofor a sequence hybridising therewith.
 23. The method according to claim22, wherein the nucleic acid sequence encodes homologues, derivatives oractive fragments of a NAP1-like protein.
 24. The method according toclaim 22, wherein said increased yield is increased seed yield.
 25. Themethod according to claim 24, wherein said increased seed yieldcomprises one or more of total weight of seeds, number of filled seedsand harvest index.
 26. The method according to claim 22, wherein thesequence of said nucleic acid is represented as SEQ ID NO: 1, or thenucleic acid encodes a NAP1-like protein as represented in SEQ ID NO: 2.27. A composition for increasing yield of a plant, comprising aNAP1-like protein or a nucleic acid encoding a NAP1-like protein. 28.The composition according to claim 27, wherein the NAP1-like protein isrepresented by SEQ ID NO: 2 or a homologue, derivative or activefragment thereof.
 29. The composition according to claim 27, wherein thenucleic acid encoding a NAP1-like protein is represented by SEQ ID NO: 1or a portion thereof or a sequence hybridising therewith, or encoding aprotein as represented in SEQ ID NO:2.